Method and apparatus for the manipulation and/or the detection of particles

ABSTRACT

Method and apparatus for the manipulation and/or control of the position of particles using time-variable fields of force; the fields of force can be of dielectrophoresis (positive or negative), electrophoresis, electrohydrodynamic or electrowetting on dielectric, possessing a set of stable points of equilibrium for the particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/996,068, filed Oct. 7, 2008, which is the U.S. national phase ofPCT/IB2006/001984 filed Jul. 19, 2006, based on BO2005A00481 filed Jul.19, 2005, the disclosures of which are each hereby incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods and apparatuses for themanipulation and/or detection of particles. The invention findsapplication principally in the implementation of biological protocols onindividual cells.

BACKGROUND OF THE INVENTION

The patent No. PCT/WO 00/69565 in the name of G. Medoro describes anapparatus and a method for the manipulation of particles via the use ofclosed dielectrophoretic-potential cages. The method described teacheshow to control the position of each particle independently of all theothers in a two-dimensional space. The force used for entrapping theparticles in suspension is negative dielectrophoresis. The individualcontrol on the operations of manipulation is carried out by programmingmemory elements and circuits associated to each element of an array ofelectrodes integrated in one and the same substrate. There follows animportant limitation due to the dimensions of each trap, limited by theneed to integrate in the space corresponding to an individual electrodethe electronics necessary for programming. Furthermore described in G.Medoro et al., 3, 317-325 (2003) IEEE Sensors Journal is an apparatusfor the manipulation of cells based upon the use of parallel elongatedelectrodes, control of which does not require the use of transistorsintegrated in the substrate itself. The shape and spatial distributionof the elongated electrodes enables creation of traps of a cylindricalshape, by means of which it is possible to entrap groups of particles.There follows an important limitation due to the impossibility in theindependent manipulation of individual particles.

Other methods for the manipulation of particles based upondielectrophoresis do not enable independent control on a multiplicity ofparticles, as described by T. Schnelle et al., Biochim. Biophys. Acta1157, 127-140 (1993). There follows an important limitation in theapplications that require the study of the interaction between amultiplicity of cells.

Other methods based upon dielectrophoresis require direct contactbetween cells and substrate, since they make use of the force ofpositive dielectrophoresis (PDEP). In particular, described in J.Suchiro, J. Phys. D: Appl. Phys., 31, 3298-3305 (1998) is a method thatenvisages the creation of traps capable of attracting to the substrate aparticle by means of forces of positive dielectrophoresis (PDEP). Theparticle consequently adheres to the substrate, from which it can bedetached and pushed towards a new region by means of an appropriatedistribution of force of negative dielectrophoresis (NDEP). In additionto the risk of causing irreparable damage to the cells, there followsome important limitations, such as for example the impossibility ofusing physiological solutions with high electrical conductivity or theimpossibility of operating with polystyrene microspheres, since in bothcases there do not exist the conditions necessary for activating theforce of positive dielectrophoresis.

Likewise, the U.S. Pat. No. 6,294,063 in the name of Becker et al.describes a method and apparatus for the manipulation of packets ofsolid, liquid or gaseous biological material by means of a distributionof programmable forces. Also in this case the contact with a surface ofreaction is a requisite indispensable for the operation of the methodand apparatus. But the biggest limitation is linked to the need for anumber of control signals (n×m) corresponding to the number ofelectrodes (n×m) if it is desired to use a passive substrate (and hencea less costly one). In order to increase the number of electrodes of theorder of many hundreds or thousands it is necessary to use an activesubstrate, as explained in P. R. C. Gascoyne et al., Lab Chip, 2004, 4,299-309, which includes transistors for addressing individually the n×melectrodes and generating locally the control signals. In this way, thenumber of input signals to the chip can be maintained within acceptablelimits.

Another known method for the manipulation of liquid particles (droplets)is electro-wetting on dielectric (EWOD), described in T. B. Jones,Journal of Micromechanics and Microengineering, 15 (2005) 1184-1187. Inthis case, an electrical field exerted by electrodes made on a substrateenables the propulsion of a droplet surrounded by a gaseous phase in adirection controlled by the sequence of energized electrodes. Devicesbased upon this principle can be obtained by including a lid (also thiscoated with a dielectric), as is taught by the patent application No. US2004/0058450A1 in the name of Pamula et al., or also simply a wirereferred to as “chain”, which establishes the electrical contact withthe droplets on top of the substrate J. Berthier et al., NSTI Nanotech2005, www.nsti.org, vol. 1, 2005. In a way similar to what has beendiscussed above regarding the use of dielectrophoresis, in order tomanipulate particles on a complete two-dimensional array via EWOD theembodiments reported in the known art resort either to a use of inputsignals corresponding to the number of electrodes of the array or to theuse of an active substrate with transistors.

A further force for the manipulation of particles is the force ofviscous friction generated by electro-hydrodynamic (EHD) flows, such aselectrothermal (ETF) flows or AC electro-osmosis. In N. G. Green, A.Ramos and H. Morgan, J. Phys. D: Appl. Phys. 33 (2000) EHD flows areused to displace particles. For example, the patent No. PCT WO2004/071668 A1 describes an apparatus for concentrating particles on theelectrodes, exploiting the aforesaid electro-hydrodynamic flows.

Other methods are known for the individual manipulation particles in atwo-dimensional space. These, however, involve the use of so-calledoptical or optoelectronic tweezers, i.e., programmable external lightsources. The result is a cumbersome and costly system, which is anundesirable characteristic in many applications. In particular A. T.Ohta et al., Tech. Dig. of the Solid-State Sensor, Actuator andMicrosystems. Workshop, 216-219, (2004) describes a possibleimplementation of said techniques.

The limitations of the known art are overcome by the present invention,which enables independent manipulation of a multiplicity of particles ina two-dimensional space, with or without contact depending upon theforces used. The implementation of the method according to the inventiondoes not require the use of electronic circuits or memory elementsintegrated in the substrate. Different embodiments of the method andapparatus according to the present invention enable manipulation ofparticles in an nm two-dimensional array of arbitrary size, with anumber of control signals of the order of n+m, or else n, or else evenwith less than ten control signals, reducing, according to differentcompromises, the parallelism and flexibility of movement of theparticles, and consequently the number of steps to perform a series ofdisplacements (a parameter that is obviously linked to the time ofexecution).

Even though the methods of the invention can be conducted withsubstrates without transistors, it is possible, however, to benefit fromthe use of active substrates to reduce the overall dimensions of theindividual elements of the array that constitute the apparatus accordingto the invention as compared to the known art or to reduce the overallnumber of the external control signals.

In addition to the possibility of manipulation of cells, the presentinvention teaches how to combine manipulation and detection byintegrating said operations on the same substrate or interfacing sensorsand actuators made on different substrates depending upon the technologyused.

SUMMARY OF THE INVENTION

The present invention relates to a method and apparatus for themanipulation of particles (in an extensive sense, as describedhereinafter) by means of time-variable non-uniform fields of force,and/or for their detection. The fields of force can be of positivedielectrophoresis or negative dielectrophoresis, electrophoresis or anyelectro-hydrodynamic motion, characterized by a set of stable points ofequilibrium for the particles (solid, liquid or gaseous). The samemethod is adaptable to the manipulation of droplets (liquid particles),exploiting effects known to the international scientific community underthe name of “electrowetting on dielectric” (EWOD). The aim of thepresent invention is to act on control of the position of each particlepresent in the sample, for the purpose of displacing said particlesindependently of one another from an initial position to any elementbelonging to the set of the final positions in a given space within amicrochamber of the device.

In a first embodiment of the method, each point of equilibrium in ahomogeneous array of elements can contain a particle or a group ofparticles. Each of said points of equilibrium can be joined withoutdistinction to any one of the adjacent points of equilibrium, allowingthe entrapped particles to share the basin of attraction thereof. Thiscontrol is made by acting exclusively on the signals shared by all theelements belonging to the same row or column, used for distributing thevoltages necessary for generation of the forces. According to thepresent invention, each path can be broken down into a succession ofelementary steps constituted by the union of adjacent basins ofattraction, thus allowing each particle to be guided from the initialposition to a final destination. Forming the subject of the presentinvention are also some practical implementations of the method by meansof apparatuses constituted by n+m+2 control signals and by n+2 m+2control signals for arrays of size n×m.

In a second embodiment of the method, the control is made by actingexclusively on the digital signals used for controlling a deviatorassociated to each element of the array, through which to distribute thevoltages necessary for generation of the forces. The object of thepresent invention is also an apparatus constituted by n+m digitalsignals for control of the distribution of the two voltages necessaryfor generation of the forces in an n×m array.

In a further embodiment of the method, each point of equilibrium in anon-homogeneous array of elements can be dedicated to containing aparticle or a group of particles (we shall call said elements “parkingcells”) or else to the transport of particles in pre-set directions (weshall call said elements “lanes” or “conveyors”). According to thepresent invention each path can be broken down into the succession ofelementary steps constituted by the entrance to, or exit from, a pre-setregion of transport, thus allowing each particle to be guided from theinitial position to any final destination.

In a further embodiment of the method, the points of equilibrium areconstrained, in groups, to moving in a synchronous way, along certainlanes. Points of exchange between the groups enable the particles topass from one group to another, i.e., to change lane. Notwithstandingthese additional constraints, the method in any case enablescarrying-out of manipulations of individual particles, and, after aseries of steps, displacement of a single particle, leaving the positionof all the others unaltered.

The object of the present invention is moreover a device thatadvantageously makes available some of the aforesaid methods,constituted by an array of electrodes, applied to which are time-variantpotentials, with or without transistors or memory elements.

The object of the present invention is also a family of apparatuses foridentification and/or quantification and/or characterization ofparticles by means of impedance meter and/or optical sensors. Thecombination of sensors and actuators is particularly useful forautomation of complex operations but proves in any case advantageous forpositioning the particles to be individuated exactly in the regions ofgreater sensitivity for the sensors (which may be integrated but alsoexternal), thus considerably improving the sensitivity of themeasurement.

DESCRIPTION OF THE INVENTION

In what follows the term “particles” will be used to indicatemicrometric or nanometric entities, either natural ones or artificialones, such as cells, subcellular components, viruses, liposomes,niosomes, microspheres, and nanospheres, or even smaller entities, suchas macro-molecules, proteins, DNA, RNA, etc., as well as drops of liquidimmiscible with the suspension medium, for example oil in water, orwater in oil, or even drops of liquid in gas (such as water in air) orbubbles of gas in liquid (such as air in water).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the principle of generation of fields of force by means ofarrays of electrodes.

FIG. 2 shows the combination of the effects due to energization ofaddressable electrodes.

FIG. 3 shows an array of addressable elements for the creation ofdielectrophoresis cages.

FIG. 4 shows the cross section of a device without transistors withaddressable nested electrodes.

FIG. 5 shows a device for the implementation of the method ofmanipulation without transistors, based upon the combination of theeffects associated to just two addressable electrodes.

FIG. 6 shows a portion of the three masks necessary for theimplementation of the prototype of apparatus with just two addressableelectrodes and an image of the prototype.

FIG. 7 shows the sequence of the elementary steps for displacement of aparticle by one step to the right in a device without transistors withjust two addressable electrodes and the experimental results.

FIG. 8 shows the sequence of the elementary steps for the displacementof a particle by one step down in a device without transistors with justtwo addressable electrodes.

FIG. 9 shows the experimental results of the manipulation of a particlealong a generic path in a device without transistors with just twoaddressable electrodes.

FIG. 10 shows the sequence of the phases of the voltages for performingthe steps of manipulation to the right or down in a device with just twoaddressable electrodes.

FIG. 11 shows a device for the implementation of the method ofmanipulation without transistors, based upon the combination of theeffects associated to the energization of four addressable electrodes.

FIG. 12 shows the sequence of the elementary steps for displacement of aparticle by one step to the right, down, to the right and to the left ina device without transistors with four addressable electrodes.

FIG. 13 shows a device for the implementation of the method ofmanipulation without transistors, based upon the combination of theeffects associated to the energization of three addressable electrodes.

FIG. 14 shows the sequence of the elementary steps for the displacementof a particle by one step to the right in a device without transistorswith three addressable electrodes.

FIG. 15 shows the sequence of the elementary steps for the displacementof a particle by one step down in a device without transistors withthree addressable electrodes.

FIG. 16 shows the sequence of the phases of the voltages for carryingout the steps of manipulation to the right or down with threeaddressable electrodes.

FIG. 17 shows a portion of the three masks necessary for theimplementation of the prototype of apparatus with three addressableelectrodes and an image of the prototype.

FIG. 18 shows a device for the implementation of the method ofmanipulation without memory elements, with just one addressableelectrode.

FIG. 19 shows the sequence of the elementary steps for the displacementof a particle by one step to the right without programming of memoryelements, with just one addressable electrode.

FIG. 20 shows an array of addressable elements for parking particleswithin dielectrophoresis cages and corridors for conveying saidparticles from one element to another of the array.

FIG. 21 shows an array of addressable elements for parking particleswithin dielectrophoresis cages and corridors, in a small number, forconveying said particles from one element to another of the array.

FIG. 22 shows a possible use of the method for selective transport ofparticles from a first microchamber to a second microchamber.

FIG. 23 shows a first apparatus for the implementation of the method ofmanipulation without transistors, with corridors and parking cells.

FIG. 24 shows a second apparatus for the implementation of the method ofmanipulation without transistors, with corridors and parking cells.

FIG. 25 shows the sequence of the phases of the voltages for carryingout the fundamental steps of the operation of the apparatus withcorridors and parking cells.

FIG. 26 shows an implementation of the method for the manipulation ofparticles with two lanes that close in a circle.

FIG. 27 shows the sequence of the steps necessary for exchange of aparticle between two lanes in the case of an array of square electrodes.

FIG. 28 shows the sequence of the steps necessary for exchange of aparticle between two lanes in the case of an array of hexagonalelectrodes.

FIG. 29 shows an apparatus for the manipulation of particles with lanesand without transistors, based upon 9 control signals.

FIG. 30 shows an apparatus for the manipulation of particles with lanesand without transistors, based upon 7 control signals.

FIG. 31 shows an apparatus for the manipulation of particles withseparate lanes and chambers, without transistors.

FIG. 32 shows an apparatus for the manipulation of particles with lanes,a completely programmable matrix array and separate chambers, withouttransistors.

FIG. 33 shows the point of exchange for the passage of a particlebetween a vertical lane and a horizontal lane of the apparatus for themanipulation of particles of FIG. 32.

FIG. 34 shows the sequence of steps necessary for exchange of a particlebetween a vertical lane of a certain group and element and a horizontallane of the apparatus for the manipulation of particles of FIG. 32.

FIG. 35 shows the operation, during the exchange between a verticalconveyor and a horizontal conveyor of a certain group and element, for avertical conveyor belonging to a different element of the same group, ofthe apparatus for the manipulation of particles of FIG. 32.

FIG. 36 shows a portion of the apparatus for the manipulation ofparticles of FIG. 32 in the immediate vicinity of the completelyprogrammable matrix array.

FIG. 37 shows the sequence of steps necessary for the passage of aparticle from the completely programmable matrix array to a waste lane.

FIG. 38 shows the point of exchange for the passage of a particle fromthe completely programmable matrix array to the auxiliary lane.

FIG. 39 shows the exchange gate for the passage of a particle from theauxiliary lane to the waste lane long.

FIG. 40 shows the exchange gate for the passage of a particle from thecompletely programmable matrix array to the exit lane.

FIG. 41 shows the exit lane of the apparatus for the manipulation ofparticles of FIG. 32.

FIG. 42 shows a circumferential stretch of the loop that surrounds theactive area of a chip constituting the apparatus for the manipulation ofparticles of FIG. 32.

FIG. 43 shows an apparatus for the manipulation of particles withouttransistors and with impedance meter sensors.

FIG. 44 shows an apparatus for the manipulation and detection and/oridentification of particles formed by a grid electrode and an array ofoptical sensors.

FIG. 45 shows the result of an experiment of manipulation and detectionobtained by means of a prototype device formed by a grid electrode andan external optical sensor.

FIG. 46 shows an apparatus for the manipulation and detection and/oridentification of particles by means of contact optical sensors andtransmitted light.

FIG. 47 shows an apparatus for the manipulation and detection and/oridentification of particles by means of contact optical sensors andtransmitted light that makes use of microlenses to increase thesensitivity of the measurement.

FIG. 48 shows an apparatus for the manipulation and detection and/oridentification of particles by means of optical sensors in which themeasurement is made by measuring portions of the array in time sequence.

FIG. 49 shows the sequence of steps for carrying out the exchangebetween a parking cell (or a conveyor) and a conveyor, in the case oflogic organization of the parking cells (conveyors) in four dimensions,for the selected parking cells (the conveyor) and for non-selectedparking cells (conveyors).

DETAILED DESCRIPTION

The aim of the present invention is the implementation of a method andan apparatus for the manipulation and/or detection of particles. By“manipulation” is meant, in particular, one of the following operationsand/or combinations thereof:

1. selection, which consists in the isolation of a given particle from asample containing a multiplicity of particles;

2. reordering, which consists in the arrangement of the particles in anorder different from the starting one;

3. union, which consists in selecting two or more particles and inbringing them closer together until they are forced against one another,for the purpose of bringing them into contact or of merging them or ofincluding them one within the other;

4. separation, which consists in separating particles that initiallywere in contact with one another.

The method is based upon the use of a non-uniform field of force (F)through which to attract individual particles or groups of particlestowards positions of stable equilibrium (CAGE). Said field can, forexample, be a dielectrophoresis field (DEP), either negativedielectrophoresis (NDEP) or positive dielectrophoresis (PDEP) field, anelectrophoretic field (EF) or else a field of electrohydrodynamic (EHD)motion, or else again electro-wetting on dielectric (EWOD).

The detection can regard one of the following aspects or a combinationthereof:

1. count of individual particles or quantification;

2. identification and/or characterization;

3. location.

In this connection the measurement of the variation of impedance and/orthe measurement of the variation of light intensity/absorbance isprincipally exploited.

Generation of Forces

There exist different methods for the generation of forces fordisplacing particles, according to the known art, by means of arrays ofelectrodes (EL) made on a substrate. Typically a cover (LID) is used,which can, in turn, be an electrode, which delimits a microchamber,within which the particles (BEAD) are typically in liquid suspension.Some schemes for the various forces are shown in FIG. 1. In the case ofDEP, the voltages applied are in-phase periodic voltages (Vphip)indicated with the symbol of addition (+) and phase-opposition periodicvoltages (Vphin) designated by the symbol of subtraction (−). By“phase-opposition voltages” are meant voltages that are 180° out ofphase. The field generates a force that acts on the particles, attractedtowards points of equilibrium (CAGE). In the case of negative DEP(NDEP), it is possible to obtain closed cages of force, according to theknown art (FIG. 1a ), if the cover (LID) is a conductive electrode. Inthis case, the point of equilibrium (CAGE) is obtained at each electrodeconnected to Vphin (−) if the adjacent electrodes are connected to theopposite phase Vphip (+) and if the cover (LID) is connected to thephase Vphin (−). Said point of equilibrium (CAGE) is normally set in theliquid at a distance from the electrodes, so that the particles (BEAD)are, in the steady-state condition, in levitation. In the case ofpositive DEP (PDEP), the point of equilibrium (CAGE) is located normallyat the surface on which the electrodes are formed (FIG. 1b ), and theparticles (BEAD) are, in the steady-state condition, in contacttherewith. For PDEP it is not necessary to have further electrodes inthe cover, because the points of equilibrium of the PDEP correspond tothe maxima of the electrical field. To manipulate particles formed bydroplets of liquid immiscible in the suspension medium and heavier thanthis (for example, water in oil), the negative dielectrophoresis(NDEPDR) can be advantageously used (FIG. 1c ) obtained by means of asubstrate (SUB) with electrodes (EL), coated by a dielectric layer (D)and by a hydrophobic layer (HPB). An array of electrodes can be used forelectrophoresis, to attract charged particles towards the electrodeswith opposite polarity. For the EHD motions, the configurations ofelectrodes generate flows that push the particles towards points ofminimum of the flow. For EWOD (FIG. 1d ), a cover (LID) containing anelectrode coated with dielectric is in general used, and the array ofelectrodes is energized by signals in phase opposition with respect tothe cover in the points in which it is desired to attract the particles(typically droplets of liquid in air). The electrodes on which theparticle must not be present are, instead, left floating. For EWOD, whenmanipulating droplets in air, on top of the array of electrodes it isalso possible to use a series of wires (FIG. 1e ) as an alternative tothe cover.

In order to describe the methods and apparatuses, for reasons ofsimplicity, in what follows use of closed cages using NDEP as force ofactuation is considered purely by way of example in no way limiting thescope of the present invention (hence it is necessary to use a coveringlid that will function as electrode). It is evident to persons withordinary skill in the sector how it is possible to generalize themethods and apparatuses described hereinafter for the use of differentforces of actuation and different types of particles.

Generation of Control on the Movement of the Particles by Means of theLogic Combination of the Effects of Force Activated by Means of Rows andColumns

In order to generate a point of stable equilibrium for the force ofnegative dielectrophoresis, it is sufficient, according to the knownart, to have available a first electrode (EL) to be supplied by means ofa signal (Vphin) in phase with the cover (LID) and one or moreelectrodes (L1) that surround completely the first electrode, suppliedby means of a signal in phase opposition (Vphip). This configuration(illustrated in FIG. 2a ) generates a minimum for the electrical field,corresponding to a point of stable equilibrium (CAGE) for the force ofnegative dielectrophoresis. Said point of equilibrium is lost if wereverse the phase of the signal applied to this first array ofelectrodes (L1), as illustrated in FIG. 2b . If we have available asecond array of electrodes (L2), such that each electrode belonging tothe second array (L2) surrounds one electrode belonging to the firstarray (L1), we shall obtain that the point of equilibrium is lost if wereverse the phase of both of the signals applied to the first array ofelectrodes (L1) and to the second array of electrodes (L2), asillustrated in FIG. 2f , in all the other cases, the cage may have adimension and shape that depends upon the voltages applied. Inparticular, in FIG. 2c and FIG. 2d we have two identical cages whilst inFIG. 2e we have one cage of larger dimensions, but centred in the sameposition. As a consequence, if we consider a multiplicity of blocks(BLOCK_i,j) each made up of an electrode (EL) and one or more arrays ofelectrodes that surround it (L1, L2), we shall find that, according tothe configuration of voltages applied to the electrodes L1 and L2 of twogeneric adjacent blocks, the following situations may arise:

a separate point of stable equilibrium for each block, the configurationof field of force of which we shall indicate with F_i;

just one point of stable equilibrium shared by the two blocks, theconfiguration of field of force of which we shall indicate with F_ii;

a separate point of stable equilibrium for each block, the configurationof field of force of which we shall indicate with F_i;

just one point of stable equilibrium shared by the two blocks, theconfiguration of field of force of which we shall indicate with F_ii;

This property can be exploited for implementation of some methods forthe manipulation of particles according to the present invention with aseries of important advantages as compared to the known art, asillustrated in what follows.

Method for the Manipulation of Particles on a Homogeneous Array withoutTransistors

An embodiment of the method according to the present invention isillustrated in FIG. 3. A homogeneous array of generic groups (BLOCK_i,j)of electrodes provide an array of attraction cages defined by points ofstable equilibrium (CAGE_i,j), each of which can entrap a singleparticle (BEAD) or group of particles. Each element (or block) of thearray (BLOCK_i,j) is electrically connected to two groups of voltages(Vrow_i[p], Vcol_j[q], p=1 . . . u, q=1 . . . v) distributed in thearray, respectively, in rows and columns and connected electrically tothe blocks that share the same row or column. The total number of rowsignals is designated by u, whilst the total number of column signals isdesignated by v.

We shall define as distance between two blocks BLOCK_i,j and BLOCK_h,kthe distance d=|i−h|+|j−k| in uniform—or Manhattan—norm, calculated onthe indices of the blocks. We shall define as “adjacent blocks” blocksthat are at a distance 1.

The same signals Vrow_i[p], Vcol_j[q] are used both for creation of thecages and for control of the position of the cages. Distributed throughthese signals are in fact the voltages necessary for the activation ofthe field of force of dielectrophoresis which have the followingproperties:

1. there always exists a configuration of potentials applied to thesignals of the array such that each attraction cage is closed anddistinct from all the others;

2. for each pair of adjacent blocks there always exists a configurationof potentials to be applied to the input signals to the pair such thatit is possible to join only and exclusively the basins of attraction ofthe pair of blocks;

3. for each pair of adjacent blocks there always exists a sequence ofpotentials to be applied to the input signals to the pair such that, ifjust one of the two attraction cages is full, it is possible to displacethe particle entrapped from one position to the adjacent one;

4. for each pair of adjacent blocks there always exists a sequence ofpotentials to be applied to the input signals to the pair such that, ifboth of the attraction cages are full, it is possible to displace bothof the particles into the same position.

The voltages to be used are generally but not exclusively periodic waves(either sinusoidal waves or else square waves) with zero mean value,chosen between a set of voltages having a different phase; by way ofnon-limiting example, it is possible to use just two phases, whichdiffer by 180° from one another.

It is evident that by joining two by two the centres of attraction ofadjacent blocks it is possible to move a particle from a generic initialposition to any final position or to bring into one and the sameposition two or more particles chosen from among all the particlespresent in the sample, without affecting the particles outside the pathof the particles undergoing movement.

The same method can be applied to the generic case of the simultaneousmanipulation of a number of particles with some restrictions. By way ofnon-limiting example, we give the restrictions for the simultaneousmanipulation of just two particles entrapped in two different cageslocated in two generic blocks:

1. if a first block and a second block are not in the same row or columnor in adjacent rows and columns, the particles entrapped in the twoblocks can be manipulated simultaneously independently of the directionand sense, provided that there are no particles entrapped in the blockscorresponding or adjacent to the row of the first block and column ofthe second block or to the column of the first block and row of thesecond block;

2. if the two blocks are on the same column but are at a distance of atleast three rows apart, they can be simultaneously manipulated in thevertical direction independently of the sense;

3. if the two blocks are on the same column but are at a distance of atleast two rows apart, they can be simultaneously manipulated in thehorizontal direction provided that the sense is the same.

4. if the two blocks are on the same row but are at a distance of atleast three columns apart, they can be simultaneously manipulated in thehorizontal direction independently of the sense;

5. if the two blocks are on the same row but are at a distance of atleast two columns apart, they can be simultaneously manipulated in thevertical direction provided that the sense is the same.

It is evident that also more than two particles can be manipulatedsimultaneously, according to the present invention, respecting for eachpair of particles the constraints listed above.

It should, however, be pointed out how, even though it is possible tomanipulate independently two or more particles that satisfy theconstraints referred to above, their simultaneous movement can have sideeffects on other cages of the array. For example, by manipulatingsimultaneously in a desired way a first particle at the block BLOCK_i,jand a second particle at the block BLOCK_h,k, an obligate movement isalso imposed on the particles of the blocks BLOCK_h,j and BLOCK_i,k. Toovercome this problem it is possible to act in different ways, dependingupon the application, via various algorithms of sequencing andserialization of the displacements, and depending upon the knowledge orotherwise of the position of all the particles.

As example we give a case of particular interest: the recovery of amultiplicity of particles from a much larger heterogeneous population.In this case, a sample is injected with particles that set themselvesrandomly on the array. Said particles can be selected, for example, atthe microscope, and, once the position of those of interest isdetermined, the problem is posed of sending them towards a gate (forexample, communicating with a second recovery microchamber), from whichthey can be made to flow out of the chip. In this case, a simple andefficient solution, which does not require the knowledge of the positionof all the particles but only of those to be selected, is the following(in the hypothesis that the gate is set on the right-hand side and atthe bottom of the microchamber):

1. Vertical virtual channels are created (routing column) in the columnsadjacent on the right to the position of each particle to be selected(selection column), freeing them from possible particles that aredisplaced onto the column further to the right (dump or waste column).

2. A horizontal virtual channel is created (routing row) at the gate ofthe recovery microchamber, freeing it from particles, as is done for thecolumns.

3. All the particles to be recovered on the routing column adjacent toeach particle are displaced.

4. The column index of the particle to be recovered furthest from therouting row is inserted into a logic set shifting-cols.

5. The row index of the particle to be recovered furthest from therouting row is defined as shifting-row index.

6. The cages in the columns belonging to the set shifting-cols and tothe row shifting-row are displaced down by a step, towards the routingrow.

7. The index shifting-row is incremented.

8. If the new row shifting-row contains particles to be recovered, thecolumn index of the new particle is inserted into the set shifting-cols.

9. If the new row shifting-row has an index lower than the onecorresponding to the routing row, the procedure returns to step 6.

Or, alternatively, after step 3 the procedure is as follows:

4′. Starting from the row furthest from the routing row, the cages ofall the routing columns are simultaneously displaced step by step down(i.e., towards the routing row), regardless of whether they containparticles or not. In this way, all the particles will be, at the end ofscanning of the entire array (corresponding to a number of steps equalto the number of rows), transferred into the routing row.

At this point, all the particles to be selected are, in known columnpositions, on the routing row.

10. The entire routing row is shifted to the right, until all theparticles have gone past the gate that communicates with the recoverymicrochamber.

11. The particles in the recovery microchamber are made to flow out ofthe chip.

Said method must be slightly complicated by preliminary operations inthe case where the distance between columns of particles to be recoveredis not always greater than 2, or in the case where there are, at thestart of the procedure, particles on the routing row that have to berecovered. For reasons of simplicity, the description of said operationsis omitted in so far as they are evident to a person with ordinary skillin the sector. Statistically, the need for carrying out thesepreliminary operations is more unlikely if the number of particles to berecovered is negligible with respect to the number of columns.

It should be noted that in general, by operating in parallel asdescribed above, the number of steps to be taken for recovery of theparticles is not significantly greater than the number of stepsnecessary with an array of totally programmable electrodes.

Apparatus for the Manipulation of Particles on a Homogeneous Arraywithout Transistors

The subject of the present invention is also an apparatus for obtainingthe field configurations necessary for the manipulation of individualparticles according to the method described previously. By way ofnon-limiting example, possible embodiments are provided, both based uponthe use of a substrate without transistors and memory elements.

Apparatus for the Manipulation of Particles with n+m+2 Control Signals

FIGS. 4 and 5 are, respectively, a cross-sectional view and a top planview of a first embodiment of the apparatus according to the presentinvention. A homogeneous array of groups (BLOCK_i,j) of electrodes formsan array of size n×m. Each block (BLOCK_i,j) is constituted by a centralelectrode (EL_i,j) connected to a signal common to the entire array(Vcore) and two concentric electrodes (ring_i,j_1, ring_i,j_2) connectedto two different voltages (Vrow_i, Vcol_j) distributed in the array,respectively, in rows and columns as illustrated in FIG. 5. A furthersignal (Vlid) is connected to the cover (LID), constituted by a singleelectrode (ITO) (illustrated only in FIG. 4). The device consequentlyrequires as a whole n+m+1+1 signals for control of n×m attraction cages,each of which can entrap a single particle (BEAD) or a group ofparticles. It is evident that a square array (n=m) minimizes the numberof control signals with respect to the number of blocks constituting then×m array.

By applying from outside a periodic voltage in phase (Vphip) to all thesignals Vrow_i and Vcol_j and a periodic voltage in phase opposition(Vphin) to the common signal Vcore and the signal Vlid connected to thecover (LID), an attraction cage (CAGE_i,j) is activated in each block(BLOCK_i,j) separated and distinct from all the others in the array. Theparticle (BEAD) entrapped in a generic block (BLOCK_i,j) can bedisplaced towards any one of the adjacent cages by means of anappropriate sequence of voltages applied to the control signals. By wayof example in no way limiting the scope of the invention, FIG. 7 showsthe sequence of the steps (a, b, c, d, e) used to displace a particlefrom the generic block (BLOCK_i,j) into the adjacent block to the right(BLOCK_i,j+1); the voltages applied to the signals involved in thevarious steps that constitute said operation are indicated in FIG. 10(sequence move_x), whilst the position of the particle in transientconditions after each step is indicated in FIG. 7 (b, c′, d′, e′, a″).Illustrated in FIG. 7 (bp, cp′, dp′, ep′, ap″), is the sequence ofimages of an experiment which correspond to the configurations (b, c′,d′, e′, a″) obtained via a prototype device.

Likewise, FIG. 8 shows the sequence of displacement of a particle in thevertical direction, from the generic block (BLOCK_i,j) into the adjacentblock downwards (BLOCK_i+1,j). The voltages applied to the signalsinvolved in the various steps that constitute said operation areindicated in FIG. 10 (sequence move_y), whilst the position of theparticle in steady-state conditions after each step is indicated in FIG.8 (b, c′, d′, e′, a″). In certain cases, it is possible to use a reducedsequence, constituted by a subset of the steps chosen from the sequencesshown in FIG. 7 and FIG. 8. Optionally, for each of the possibledirections, it is possible to use a sequence constituted by stepsdifferent from the ones described by way of non-limiting example inFIGS. 7 and 8.

It is evident that any path that starts from a generic position in thearray and terminates in any other position of the array can be brokendown into the succession of the elementary steps illustrated in FIGS. 7and 8 and in the analogous steps in the opposite direction. A practicalexample of said concept is illustrated in FIG. 9, which shows thesuccession of the elementary steps to displace a polystyrene microspherefrom the initial position (BLOCK_i,j) towards the destination(BLOCK_i+1,j+4) following a generic path.

Implementation of the apparatus according to the present invention canbe obtained by exploiting different technologies according to the knownart. Shown by way of example in no way limiting the scope of the presentinvention in FIG. 6a-c are the masks necessary for a possible embodimentof the apparatus by means of photolithographic techniques according tothe known art and shown in FIG. 6d is an image of the prototype. Threemasks and two metal levels are sufficient for the implementation. Theminimum distance (PITCH) between the centres of two adjacent blocks is 5times the pitch between surface metallizations. In this device the pitch(PITCH) is 100 μm; this means that the technology required forfabrication must enable the production of electrodes the minimum pitchof which is 20 μm. For production of the electrodes noble metals (gold,platinum, etc.) can be used, or else conductive oxides, which areparticularly useful in the case where said oxides are transparent(Indium Tin Oxide—ITO). For the production of the substrate insulators(glass, polycarbonate, etc.) can be used, or else semiconductors(silicon, etc.), in which case a passivation oxide is required forinsulating the substrate electrically from the first metal level. Forthe production of the cover (LID), an insulating substrate can be usedprovided that it is equipped with an electrode which also may be madewith metals or conductive oxides, which are particularly useful in thecase where said conductive oxides are partially or totally transparent.Likewise, semitransparency can be obtained using a non-transparent metalin the form of a grid.

It is evident to persons with ordinary skill in the sector, that othergeometries different from the ones described in the present patent byway of example, can be used for the production of the apparatusaccording to the present invention. By way of non-limiting example, wemay cite electrodes with circular, hexagonal, rectangular geometries,etc. Likewise, it is evident that other materials, different from theones referred to in the present patent, can be used for the productionof the apparatus according to the present invention. By way ofnon-limiting example we may cite materials such as aluminium, titanium,tantalum, gold, etc.

Apparatus for the Manipulation of Particles with 4n+4 m+2 ControlSignals

FIG. 11 is a top plan view of a different embodiment of the apparatusaccording to the present invention. In this case, four signals are usedfor each row and four signals for each column, plus a global signalVcore common to all the blocks (distributed herein by column) and asignal Vlid. The external and internal ring electrodes of each block,are divided into two, vertically and horizontally, respectively.Alternately connected to the electrodes of each block are just two ofthe four row signals, and just two of the four column signals. The rowsignals and column signals are normally all connected to Vphip, andgenerate a field configuration (F_i), with an attraction cage(CAGE_i,j), for each block. By connecting to Vphin seven signals chosenappropriately from among the control signals by rows and columns, it ispossible to generate a second configuration (F_ii), which joins theattraction cages of two adjacent blocks. As illustrated in FIG. 12 it isthus possible to displace a particle (BEAD) to the right (R), to theleft (L), downwards (D) or upwards (U), without altering the position ofthe other particles possibly entrapped in nearby cages, simply byapplying the field configuration (F_ii) and then the initial fieldconfiguration (F_i) again.

As compared to the embodiment with n+m phases, this embodiment presentsthe advantage of requiring only two field configurations for eachelementary displacement, and the disadvantage of requiring a number ofcontrol signals four times greater.

Apparatus for the Manipulation of Particles with n+2 m+2 Control Signals

FIG. 13 is the top plan view of a further embodiment of the apparatusaccording to the present invention. A homogeneous array of blocks(BLOCK_i,j) forms an array of size n×m. Each block (BLOCK_i,j) is madeup of: a central electrode (EL_i,j) connected to a signal common to theentire array (Vcore); an L-shaped electrode (elle_j) connected tosignals distributed in the array according to columns (Venable_j); andtwo electrodes, one in the form of a vertical segment (wallx_i) and theother in the form of a horizontal segment (wally_i) connected to twodifferent signals (Vrow_i[x], Vrow_i[y]) distributed in the arrayaccording to rows and arranged radially on the outside (with respect tothe central electrode) of the electrode elle_j. A further signal (Vlid)is connected to the cover (LID), constituted by a single electrode(ITO). The device consequently requires as a whole n+2 m+1+1 signals forcontrolling n×m attraction cages, each cage being able to entrap asingle particle (BEAD) or a group of particles. It is possible to showthat a rectangular array where n=2m minimizes the number of controlsignals with respect to the number of blocks constituting the array(n×m).

By applying from outside a periodic voltage in phase (Vphip) to all thesignals Vrow_i[x], Vrow_i[y] and Venable_j and a periodic voltage inphase opposition (Vphin) to the common signal Vcore and to the signalVlid connected to the cover (LID), an attraction cage (CAGE_i,j) in eachblock (BLOCK_i,j) separate and distinct from all the others in the arrayis activated. The particle (BEAD) entrapped in each generic block(BLOCK_i,j) can be displaced towards any one of the adjacent cages bymeans of an appropriate sequence of voltages applied to the controlsignals. By way of example in no way limiting the scope of theinvention, FIG. 14 shows the sequence of the steps (a, b, c, d) used todisplace a particle from the generic block (BLOCK_i,j) into the adjacentblock to the right (BLOCK_i,j+1); the voltages applied to the signalsinvolved in the various steps of said operation are indicated in FIG. 16(sequence move_x), whilst the position of the particle in transientconditions after each step is indicated in FIG. 14 b′, c′. Likewise,FIG. 15 shows the sequence of the steps (a, b, c, d) used to displace aparticle from the generic block (BLOCK_i,j) into the adjacent blockdownwards (BLOCK_i+1,j). The voltages applied to the signals involved inthe various steps that make up said operation are indicated in FIG. 16(move_y), whilst the position of the particle in steady-state conditionsafter each step is indicated in FIG. 15 b′, c′. In certain cases, areduced sequence can be used, made up of a subset of the steps chosenfrom the sequence illustrated in FIG. 14 and FIG. 15. Optionally, foreach of the possible directions, a sequence can be used consisting ofsteps different from the ones shown by way of non-limiting example inFIG. 14 and FIG. 15.

It is evident that any path that starts from a generic position in thearray and terminates in any other position of the array can be brokendown into the succession of the elementary steps illustrated in FIG. 14and in FIG. 15, and in the analogous steps in the opposite direction.

The implementation of the apparatus according to the present inventioncan be obtained exploiting different technologies according to the knownart. By way of example in no way limiting the scope of the presentinvention, shown in FIG. 17 (a-c) are the masks necessary for a possibleimplementation of the apparatus by means of photolithographic techniquesaccording to the known art, and shown in FIG. 17d is an image of theprototype. Three masks and two metal levels are sufficient for theimplementation. The implementation of the apparatus according to thepresent invention can be obtained exploiting different technologiesaccording to the known art. The pitch (PITCH), i.e., the distancebetween the centres of two adjacent blocks, in this device is 100 μm.For the electrodes, noble metals (gold, platinum, etc.) can be used orelse conductive oxides, which are particularly useful in the case wheresaid oxides are transparent (Indium Tin Oxide—ITO). For the substrateinsulators (glass, polycarbonate, etc.) can be used or elsesemiconductors (silicon, etc.), in which case a passivation oxide isrequired for electrically insulating the substrate from the first metallevel.

Method for the Manipulation of Particles on a Homogeneous Array withoutMemory Elements

A further embodiment of the method according to the present inventionuses an array of attraction cages (CAGE_i,j), in which each block(BLOCK_i,j) is electrically connected to two groups of signals(Vrow_i[p], Vcol_j[q]) distributed in the array, respectively, in rowsand columns. Some of these signals are used for the distribution of thevoltages (Vphin, Vphip) necessary for creation of the cages (CAGE),whilst others are digital signals used for control of the phase to beapplied to the electrodes. In this case, the position of the points ofstatic equilibrium (CAGE_i,j) is controlled by means of electroniccircuits, which determine for each block whether the attraction cage isin isolation or connected to adjacent cages.

Apparatus for the Manipulation of Particles on a Homogeneous Arraywithout Memory Elements

The subject of the present invention is also an apparatus for theproduction of the field configurations necessary for the manipulation ofindividual particles according to the method described previously. Byway of example, a possible embodiment is shown based upon the use ofactive substrates, in which, however, each block is without memoryelements, unlike what is reported in the known art.

FIG. 18 is a top plan view of a possible embodiment of the apparatusaccording to the present invention. A homogeneous array of blocks(BLOCK_i,j) forms an array of attraction cages of size n×m. Each block(BLOCK_i,j) is constituted by a central electrode (EL_i,j) connected toa signal common to the entire array (Vphin) and an electrode (ring_i,j)connected to the output of a multiplexer, which receives at input twodifferent signals (Vphin, Vphip) and the output of which depends uponthe logic combination of row digital control signals (row_i) and columndigital control signals (col_j) according to the following table oflogic values:

TABLE-US-00001 row i = 0 Row i = 1 col j = 0 Vphip Vphip col j = 1 VphipVphip

A further signal (Vlid) is connected to the cover (LID), not shown,constituted by a single electrode (ITO). The device consequentlyrequires as a whole two analog signals (Vphin and Vphip) and n+m digitalsignals for controlling n×m attraction cages, each of which can entrap asingle particle (BEAD) or a group of particles. It is evident that asquare array (n=m) minimizes the number of control signals with respectto the number of blocks constituting the array (n×m).

By applying a logic value 0 to all the signals row_i and col_j and aperiodic voltage in phase opposition (Vphin) with respect to the signalVlid connected to the cover (LID), an attraction cage (CAGE_i,j) isactivated in each block (BLOCK_i,j) separate and distinct from all theother in the array. The particle (BEAD) entrapped in each generic block(BLOCK_i,j) can be displaced towards any of the adjacent cages by meansof an appropriate sequence of logic values applied to the controlsignals. By way of example in no way limiting the scope of theinvention, FIG. 19 shows the sequence of the steps (a, b, c,) used todisplace a particle from the generic block (BLOCK_i,j) into the adjacentblock to the right (BLOCK_i,j+1); the sequence of the logic valuesapplied to the signals row i, col j and col j+1 is the following:

TABLE-US-00002 (a) (b) (c) (a′) col j 0 1 0 0 col j + 1 0 1 1 0 Row i 01 1 0The position of the particle in transient conditions after each step isindicated in FIG. 19 b′, c′, a′.

It is evident that the method applies in a similar way for anydirection. In addition, any path that starts from a generic position inthe array and terminates in any other position of the array can bebroken down into the succession of the elementary steps constituted bydisplacements of just one position. The implementation of the apparatusaccording to the present invention can be obtained exploiting differenttechnologies of fabrication of microelectronic circuits according to theknown art.

Method for the Manipulation of Particles with Lanes and Parking Cells

A further embodiment of the method according to the present invention isillustrated schematically in FIG. 20. The method uses a set of points ofstable equilibrium that are static for the force (F) that acts on theparticles, located within blocks (BLOCK_i,j), the function of which isthat of entrapping stably a particle, and a set of points of stableequilibrium moving along lanes in the horizontal direction (HRCH1-HRCHM)or vertical direction (VRCH1-VRCHN). Each of these blocks (BLOCK_i,j)can be configured for entrapping the particle or pushing it within thebasin of attraction of one of the points of stable equilibrium movingalong the lanes. This can be obtained exploiting one of the methodsdescribed according to the present invention, for example joining thepoint of stable equilibrium of the block to one of the points of stableequilibrium of the lanes. It is evident that each of the particlespresent in the sample can consequently be parked within the blocks orelse can be displaced from one block to any other one exploiting one ormore lanes, in the most convenient direction. The particle can, in fact,enter a lane in motion and, likewise, the particle can exit from theselanes to enter a new block, or to change the direction of motion,passing onto a new lane. It is evident to persons with ordinary skill inthe sector that each particle can pass from one block to any other oneexploiting the method according to the present invention. The advantageof this technique consists in a reduction in the total number of signalsdedicated to control of the entire array as is illustrated in theapparatuses described in what follows. Likewise, FIG. 21 shows a secondembodiment of the method with a reduced number of horizontal paths. Itis evident that also in this case each particle can pass from one blockto any other one exploiting the single horizontal path (HRCH1). Thistechnique enables a further reduction in the number of the signalsrequired and increases the surface useful for providing cages. By way ofexample in no way limiting the scope of the present invention, FIG. 22shows a possible application of the method. Present inside amicrochamber (CHW) is an array of blocks (BLOCK_i,j) the function ofwhich is the one described previously. The microchamber splits the arrayinto two parts: one part (MCH) provided for containment of the sample tobe processed, the other (RCH) provided for containment of the processedsample. For example, this scheme could be used for selecting just oneparticle retained in the first microchamber ((MCH) and recover it fromthe second microchamber (RCH). Each block (BLOCK_i,j) is functionallyconnected to a vertical corridor (VRCHJ), the direction and sense ofmovement of which is coherent within the entire array and terminateswith a single horizontal corridor (HRCH1), the direction and sense ofmovement of which is chosen so that the particles conveyed can betransferred from the first microchamber (MCH) to the second microchamber(RCH) and then be accumulated in a single area through a final corridor(VRCHR). The selection of a particle from among the n×m retainedinitially in the first microchamber can be made, for example, bytransferring it onto the corresponding lane and conveying it into thesecond microchamber (RCH), initially free from particles, from which theparticle selected can be extracted.

Apparatus for the Manipulation of Particles with Lanes and Parking Cellswithout Transistors

The subject of the present invention is also an apparatus for theproduction of the field configurations necessary for the manipulation ofparticles according to the method described previously, based upon theuse of parking blocks and lanes. By way of non-limiting example, apossible embodiment is shown based upon the use of passive substrates,in which each block is without any memory elements or transistors.

FIG. 23 is a top plan view of a first embodiment of the apparatusaccording to the present invention. A homogeneous array of blocks(BLOCK_i,j) forms an array of attraction cages capable of entrapping aparticle stably. Each block (BLOCK_i,j) is made up of: a centralelectrode, connected to a control signal (Vcage_j) common to all theblocks of the same column (or even to the entire array); a set ofelectrodes connected to a signal (Vpj) common to the entire array, andcorresponding to Vphip; an electrode connected to a control signal(Vcol_j) common to all the blocks of the same column; and, finally, anelectrode connected to a control signal (Vrow_i) common to all theblocks of the same row. By acting on the phase applied to the signalsVcage_j, Vcol_j and Vrow_i, the point of stable equilibrium for theforce that entraps the particle can be displaced from the block(BLOCK_i,j) towards a corridor (VRCHJ) or from the corridor towards theblock. Each corridor is made up of an array of electrodes connected tosignals common to the entire corridor (V1_j, V2_j and V3_j). By actingon the phase applied to these signals it is possible to create and todisplace as desired points of stable equilibrium for the force F alongthe entire corridor. Likewise, the apparatus can have available one ormore corridors oriented in a horizontal direction (HRCH), controlled bythree signals common to the entire corridor (Vh_1, Vh_2 and Vh_3),operation of which is altogether similar to that of the corridorsoriented vertically (VRCHJ). FIG. 25 shows the voltages applied to thesignals involved in the various steps that constitute the sequence forexit from a block (CHACCIJ), for shifting by a position along thevertical corridor (CONVEYV), for entry into the horizontal corridor(HCHACC), and for running along the horizontal corridor (CONVEYH). It isevident that in order to reverse the sense of travel along thehorizontal corridor or vertical corridor it is sufficient to reverse thesequence of the phases with respect to that illustrated in FIG. 25.

FIG. 24 shows the top plan view of a further embodiment of the apparatusfor the manipulation with lanes and parking cells according to thepresent invention. Operation is altogether similar to that of theembodiment described previously, but enables a greater density ofattraction cages to be obtained per unit surface in so far as, for eachcolumn of blocks BLOCK_ij, the n electrodes at potential Vpj arereplaced by a single comb-shaped electrode at potential Vpj.

The number of control signals for both of the implementations withouttransistors of the apparatus with lanes and parking cells, for an arrayof n×m blocks with a number of independent horizontal and verticalcorridors equal to g and f respectively, is 2n+m+3(g+f)+2. If the signalVcage_j is shared among all the columns, the number of signals drops ton+m+3(f+g)+2. Typically (as illustrated in the examples), f=m, but it ispossible also to share the same vertical lane between two columns ofcages, in which case f=m/2. The number of horizontal channels can bechosen as desired. The greater the number of horizontal channels, thegreater the flexibility, but the smaller the useful area for the cagesand the greater the number of control signals required.

In practice, in the example described above, the parking cells arelogically organized in a two-dimensional (row, column) space, and eachhave access to a vertical lane when the signals of each of thedimensions (row and column) are activated, in the appropriate sequence.According to the present invention, it is also possible to achievedifferent compromises between the number of control signals and thesurface necessary for the transfer of a cage from a parking cell to alane, by logically organizing the aforesaid parking cells in a number ofdimensions higher than two. In fact, the surface dedicated to thetransfer from the parking cell to the lane is proportional to the numberof logic dimensions (this area is to be considered wasted). Theadvantage is that the number of parking cells corresponds to the productof the number of control signals by each dimension. By way of example,10,000 parking cells can require 100 rows and 100 columns, i.e., 200control signals, in the case of two dimensions or else 22*3=66 controlsignals, in the case of three dimensions, or 10*4=40 control signals,for organization in four dimensions. The spatial arrangement of theparking cells, can remain obviously two-dimensional, whatever the logicorganization.

The transfer of the cage from the parking cell to the lane is made ingeneral by means of an appropriate sequence of activation of the controlsignals. The sequence is chosen so as to push from the parking cell tothe lane only the cage that corresponds to the desired location, whilstall the other cages in parking cells make at most a few steps in thedirection of the lane, but reverse then the sense of displacementwithout completing the transfer, and at the end drop back into theoriginal position. FIG. 49 shows the example of a possible sequence ofactivation of the electrodes (EL), to bring particles (BEAD) from aparking cell (cage) to a conveyor (cony), in the case of a logicorganization in four dimensions (d1, d2, d3, d4). The underlining ofeach signal “di” symbolizes the fact that the cell corresponds to aselected dimension. Consequently, the signals di are programmable bothfor the negative phase (activation, indicated by a shading) and for thepositive phase (empty). The signal cage is in this case programmable,and the number of different signals cage must correspond to the numberof addressing signals (D1) of the first dimension d1. In this way, themovement of the particle (BEAD) from the starting cage is repeatable anddeterministic, as illustrated in FIG. 49. The number of parking cellsaddressable with D dimensions is equal to the product of the number ofaddressing signals of each dimension, i.e., D1×D2× . . . D_(D), whilstthe number of necessary control signals amounts to 2×D1+D2+ . . .+D_(D).

The implementation of the apparatus according to the present inventioncan be obtained exploiting different technologies according to the knownart. By way of example, we may cite photolithographic techniques. Threemetal levels are ideal for minimizing the resistance of the paths, in sofar as in this case for the row and column lines it is not necessary tohave any transition between one level and the other (the ways and theassociated resistances are avoided). Two metallizations are, however,sufficient in the case where ways are also used for the row and columnsignals. The horizontal and vertical pitches (PITCH), i.e., the distancebetween the centres of two adjacent blocks either horizontally orvertically, in this device is equal, respectively, to five times ortwice the pitch between adjacent surface metals. To obtain theelectrodes noble metals (gold, platinum, etc.) can be used or elseconductive oxides, which are particularly useful in so far as saidoxides are transparent (Indium Tin Oxide—ITO). To make the substrateinsulators (glass, polycarbonate, etc.) or else semiconductors (silicon,etc.) can be used. To make the cover (LID) an insulating substrateprovided with an electrode can be used, which can also be obtained bymeans of metals or conductive oxides, which are particularly useful whensaid conductive oxides are partially or totally transparent. It isevident to persons with ordinary skill in the sector that othergeometries different from the ones described in the present patent byway of example can be used for the production of the apparatus accordingto the present invention.

In general, apparatuses with arrays of regular electrodes (i.e., thosewithout rings or the like) are preferable in the use with the EWODforce.

Apparatus for the Manipulation of Particles with Lanes and Parking Cellswith Transistors and/or Memory Elements

By way of non-limiting example, a further possible embodiment is shownbased upon the use of active substrates, in which transistors and/ormemory elements are used.

Apparatus for the Manipulation of Particles with Conditioning Circuitsfor Lanes

Each of the signals (Vh_1, Vh_2, Vh_3) used for supplying the electrodesof the corridors oriented horizontally (HRCH), and each of the signals(V1_j, V2_j and V3_j) used for supplying the electrodes of the corridorsoriented vertically (VRCHJ) can be connected to signals common to theentire apparatus (Vphin, Vphip) through electronic circuits that formmultiplexers. Said multiplexers can be programmed through digitalsignals or by means of individually addressable memory elements. Thecircuit embodiment that implements this scheme can be obtained accordingto any of the methods known to persons with ordinary skill in thesector. This technique enables a reduction in the total number ofsignals necessary for driving and/or programming the entire apparatus.

Apparatus for the Manipulation of Particles with Conditioning Circuitsfor Parking Cells

Likewise, each of the signals (Vcage_j, Vcol_j, Vrow_i) used forsupplying the electrodes of the parking cells can be connected tosignals common to the entire apparatus (Vphin, Vphip) through electroniccircuits that form the multiplexers. Said multiplexers can be programmedthrough digital signals or by means of individually addressable memoryelements. The circuit embodiment that implements this scheme can beobtained according to any of the methods known to persons with ordinaryskill in the sector. This technique enables a reduction in the overallnumber of signals necessary for driving and/or programming the entireapparatus.

Method for the Manipulation of Particles with Lanes

In a further embodiment of the method according to the present inventionthe points of equilibrium are constrained, in groups, to move in asynchronous way, along pre-set paths referred to as “lanes”. Points ofexchange between the groups enable the particles to pass from one groupto another, i.e., to change lane. Notwithstanding these additionalconstraints, the method enables in any case manipulations of individualparticles, and, after a series of steps, displacement of a singleparticle, leaving the position of all the others unaltered.

An example of the working principle of the method is illustrated in FIG.26. Two lanes, that are closed in a circle, are sufficient. In the firstlane (C_STORE), driven by NS phases S₁·S_(NS), repeated NIS times,particles can be introduced, possibly even in a random order. Bytransferring one or more particles onto the second lane (C_TMP), drivenby NT phases T₁·T_(NT), repeated NIT times, it is possible to re-orderthe particles on the first lane. The minimum number of phases for eachlane is 3. Consequently, with 6 phases it is possible to control anarbitrary distribution of particles. The exchange between two lanes canbe obtained with a sequence of steps as illustrated in FIG. 27a-e ,which shows a particle (BEAD) in an attraction cage (CAGE) on a firstlane (CON_1) whilst it is being carried into the point of exchange withthe other lane [FIG. 27c ], by changing the programming of theelectrodes (EL). By moving away [FIG. 27d ] the cage of the first lanewhen a cage is present in the point of exchange on the second lane(CON_2), the particle passes onto the latter. FIG. 28 shows a similarsequence in the case of an array of hexagonal electrodes. Thisembodiment is particularly suited to use with the EWOD force. However,it is possible to obtain the exchange between lanes using more complexconfigurations of electrodes, exploiting one of the methods describedfor the purposes of the present invention.

In a further embodiment of the method according to the presentinvention, just a single lane is used for causing all the particles toshift in order to reposition a given particle in a given position. It isevident that said method applies to the generic case of a number oflanes, without, however, envisaging any exchange between lanes. In thiscase, it is useful for the lanes not to be constrained to one another.

Apparatus for the Manipulation of Particles with Lanes withoutTransistors

Apparatus for the Manipulation of Particles with 9 Control Signals

FIG. 29 shows a preferential embodiment of an apparatus for themanipulation of particles with lanes, without the use of transistors.NCV vertical circular lanes VC_1. VC_NCV each form NI cages (CAGES), bymeans of 3 phases, V1, V2 and V3. Said phases are connected repeatedlyat each iteration I_1 . . . I_NI of a group of 3 electrodes. Said phasesare common to all the lanes. A second horizontal circular lane (HCONV),driven by three phases H1, H2 and H3, comprises NCV points of exchangewith the vertical conveyors, active in the phase V1+H1, so that it ispossible to transfer simultaneously the contents of NCV cages from thevertical lanes to the horizontal lane. Said vertical and horizontallanes are obtained in a first microchamber (MCH). A third lane (RCONV),driven by the phases R1, R2 and R3, is obtained in a second microchamber(RCH), separated from the first by a diaphragm (CHW). Said third lanecomprises a point of exchange active during the phase H2+R2.

This apparatus is particularly suitable, for example, for isolatingindividual particles, for example cells suspended in a liquid. Amultitude of particles can be injected into the first microchamber(MCH). A liquid without particles is injected in the second microchamber(RCH). One or more particles of interest can be selected and conveyedfrom the vertical lanes of the first microchamber (MCH) to thehorizontal lane and from here to the third lane in the secondmicrochamber. From here the particles can be made to flow out andrecovered separately.

Apparatus for the Manipulation of Particles with 7 Control Signals

The embodiment of the apparatus can be further simplified, for theisolation of individual particles, by constraining the third lane(RCONV) to move in a synchronous way with the vertical lanes so as toshare the phases V1, V2 and V3 thereof, as illustrated in FIG. 30. Inthis case, however, a phase (THR) is to be added for controlling thetransfer from the horizontal conveyor (HCONV) to the third conveyor(RCONV). The total number of phases is thus reduced to 7.Notwithstanding the constraints of this apparatus with the use of lanesalone, it should be noted that the number of steps to bring anindividual particle from a point of the first microchamber (MCH) into apoint of the second microchamber (RCH) is, to a first approximation,approximately equal to the number of steps of an apparatus that enablesindependent movement of all the particles.

Apparatus for the Manipulation of Particles with Separate Lanes andChambers

A preferential embodiment of the apparatus for management of particlesof a different type is shown in FIG. 31. In this case, each verticallane is obtained in a separate microchamber and is controlled byseparate signals. For example, in different vertical chambers differentparticles can be injected. It is thus possible to transfer in an orderlyway, onto the horizontal lane (HCONV), particles of different types, orelse it is possible to bring particles of one type to interact withparticles of a second type coming from a second microchamber.

Apparatus for the Manipulation of Particles with Lanes with Transistorsand/or Memory Elements

Each of the signals used for supplying the electrodes of the corridors(C_STORE, C_TEMP, VC_i, HCONV, RCONV) can be connected to signals commonto the entire apparatus (Vphin, Vphip) through electronic circuits thatform multiplexers. Said multiplexers can be programmed through digitalsignals or by means of individually addressable memory elements. Thecircuit embodiment that implements this scheme can be obtained accordingto any of the methods known to persons with ordinary skill in thesector. This technique enables reduction in the overall number ofsignals necessary for driving and/or programming the entire apparatus.

Apparatus for the Manipulation of Particles with Lanes and CompletelyProgrammable Array

In a further embodiment of the present invention, the technology of theapparatus of FIG. 23, implemented, according to techniques similar tothose used in the apparatuses of FIGS. 29, 30, 31 already described, isused to obtain the complex apparatus illustrated in FIG. 32, whichenables optimization of the possibilities and times of manipulation ofthe particles and at the same time containment of the number of controlsignals necessary. According to what is illustrated in FIG. 32, thisapparatus according to the invention is divided by a diaphragm (CHW)made of polymeric material into two microchambers (MCH, RCH).

The first microchamber (MCH) is substantially constituted by:

a. a first multiplicity and a second multiplicity of vertical circularlanes (definable also as “conveyors”) (i.e., ones forming a closed loop,albeit elongated) VC1_1 . . . VC1_NCV, and VC2_1 . . . VC2_NCV eachforming NI cages (CAGES), by means of three phases V1, V2 and V3,connected repeatedly at each iteration I_1 . . . I_NI of a group ofthree electrodes;

b. a first horizontal circular lane and a second horizontal circularlane HCONV_UP, HCONV_DOWN (or even simply a linear lane, i.e., withelectrodes arranged to form a portion of array in a straight lineinstead of in a loop), driven by four phases H1, H2, H3 and H4comprising NCV points of exchange with the vertical lanes (conveyors),active in the phase V2+H3, so that it is possible to transfersimultaneously the contents of one or more cages from the vertical lanesto the first horizontal lane;

c. a third circular horizontal (or simply linear) lane HCONV_AUX, drivenby four phases AUX1, AUX2, AUX3 and AUX4, which comprises NCAUX1 pointsof exchange with the conveyor or an upper horizontal lane HCONV_UP andan identical number NCAUX2 of points of exchange with the conveyor orlower horizontal lane HCONV_DOWN, situated in positions corresponding toone another;

d. a completely programmable matrix array of electrodes, for example asquare array of 5×5 electrodes, each controlled individually throughspecial dedicated phases, or yet again using electrodes of a completelyactive type, as in the known art, each equipped with programmable memoryelements and transistors, so as to form in use a matrix array ofindividually programmable attraction cages;

e. a first circular vertical dump lane VCW_UP and a second circularvertical dump lane VCW_DOWN driven by 3 phases in a way substantiallysimilar to what has already been described for the vertical lanes VC1_iand VC2_j, which have the function of removing undesired particles fromthe array;

f. a long circular vertical dump lane VCW_LONG, having a dimensionapproximately twice that of the vertical lanes VC1_i and VC2_j, which isalso driven by three phases in a way substantially similar to what hasalready been described for the vertical lanes VC1_i and VC2_j, set inthe portion of the microchamber MCH on the side opposite to the array.

The second microchamber (RCH) is substantially constituted by an exitlane RCONV, driven by the four phases R1, R2, R3 and R4, for conveyingthe particles leaving the array of interest into the secondmicrochamber, through a discontinuity of the diaphragm made of polymericmaterial CHW constituting a passage of communication between the twomicrochambers. There is moreover provided a horizontal feedback laneHCONV_FB, driven by four phases FB1, FB2, FB3 and FB4, lyingsubstantially on the same straight line identified by the auxiliaryhorizontal lane, by means of which it is possible to bring a particleback from the exit lane RCONV, and hence from the microchamber RCH, intothe array, once again through the aforesaid passage in the diaphragmCHW.

In a particular embodiment of the present invention, the verticalcircular lanes are 400, arranged in 20 groups of 20 elements. Since thefirst microchamber MCH is fundamentally divided by the horizontal lanesinto two half-chambers, a top one and a bottom one, the vertical lanesare 200 in the top half and 200 in the bottom half. The structure ishence completely symmetrical.

Each individual lane is able to displace a particle and rotate it usinga three-phase protocol. It is possible in any case to extract from alane a particle of interest using one of the (controllable) NCV pointsof exchange, positioned immediately on top of the horizontal laneHCONV_UP for the vertical lanes upwards and immediately underneath thehorizontal lane HCONV_DOWN for the vertical lanes downwards. Each pointof exchange is defined by a pair of electrodes, referred to,respectively, as “element” and “group” (FIG. 32). Since the groupelectrodes and element electrodes are 20, the number of the addressableexchanges is 400, equal to the number of the vertical lanes. Thespecial-phase signal is the same for each lane within a group, so thatthe behaviour of the signal special phase and of the signals of a groupelectrode are the same for each conveyor of the group. The same does notapply to element electrodes. It is always possible, in this way, totransfer a particle of interest from a vertical conveyor to a horizontalconveyor in order to convey it as far as the programmable array andpossibly to the exit point, without loading any other particle into thehorizontal conveyor. The change of direction, i.e., the transfer from avertical lane to a horizontal lane is made possible by an electrodeguided by a special phase (FIG. 33). Usually, said electrode is locatedin the same phase as the signal of Phase 2, but in the case of aparticle of interest, when all the other signals of Phase 2 remainnegative (i.e., active) also the special signal becomes negative (FIG.34), thus leaving the cell to be transferred through the point ofcontact if the element electrode and group electrode are active. When,instead, the signal of the element is not in the negative phase (what isinstead true for all the other 19 lanes not concerned in the exchange),the operation is the one illustrated in FIG. 35.

In this way, the particles can be joined to the conveyors upwards anddownwards. The operation illustrated in FIG. 35 is moreover useful whenit is desired to modify the order of the particles within one and thesame vertical lane, in so far as it makes possible temporary deposit ofa particle at the group electrode external to the vertical lane, andthen get it to come back into the vertical lane itself.

In a way similar to what was illustrated previously in the descriptionof the apparatus with lanes and parking cells without transistors, alsoin this case of the apparatus with lanes and programmable array it ispossible to adopt a logic organization of the conveyors not in twodimensions (as described above) but in D dimensions. By way of example,reference may be made once again to FIG. 49, described previously, asrepresentation of the transfer of a particle (BEAD) from the end of avertical conveyor (cage) to a horizontal conveyor (cony). To personswith ordinary skill in the sector it is clear how it is possible togeneralize the sequence of operations for performing the exchanges fromvertical lanes to the horizontal lane only for the lane selected, i.e.,only for that for which all the D (=4, in the example) signals ofexchange of each dimension are selected.

With the horizontal lanes (HCONV_UP, HCONV_DOWN), the particles ofinterest can be transferred into the completely programmable matrixarray, in which it is possible to carry out complex operations, such asfor example the division of clusters of particles. This is particularlyuseful, for example, when the mean density of cells per cage in thesample injected is equal to or greater than one. In this case, theprobability of having a single cell in the cage decreases, andconsequently it is likely for the cells of interest to form part of acluster. The presence of the completely programmable matrix arrayenables segregation in different cages of the cells forming part of acluster.

In the preferential embodiment, a matrix array is a square of 5×5completely programmable electrodes, as illustrated in FIG. 36, whichshows the relative interaction thereof with the horizontal conveyors andauxiliary conveyor.

By means of the matrix array it is possible to select and withhold theparticles of interest, whilst, after the segregation in separate cages,the others can be moved away after being transferred to the dump lanes.The points of exchange between array and dump lanes function like theother points of exchange, but without the two element and groupelectrodes (FIG. 37).

The auxiliary lane HCONV_AUX can be used as support for the twohorizontal lanes HCONV_UP and HCONV_DOWN, for example in the case of anymalfunctioning due to clogging of particles, etc. In a preferredembodiment of the apparatus, between the three horizontal lanes 12points of exchange are provided, made with a double point of exchange,as illustrated in FIG. 38.

The auxiliary lane can also be used to eliminate the undesiredparticles, particularly during the step of start-up of the apparatus.FIG. 39 shows the left-hand end of the auxiliary lane, where it ispossible to transfer the particles in the long dump lanes through anindividual point of exchange.

Located at exit from the matrix array (FIG. 40) are the dump laneupwards VCW_UP and the dump lane downwards VCW_DOWN. The exit lane RCONVis a 4-phase lane, which conveys the cells of interest out of themicrochamber MCH and into the microchamber RCH. In order to have thehighest possible number of cages, the path towards the exit pointproceeds by zigzagging (FIG. 41) all the way through the microchamberRCH. When the particles are in the exit lane, they can be brought backinto the array by means of the horizontal feedback lane HCONV_FB.

The latter splits the exit lane in a symmetrical way, identifying ineffect a top exit half-lane and a bottom exit half-lane. It should benoted that said half-lanes are completely independent of the operativestandpoint, and it is consequently possible to use even just one ofthem.

The active area of the apparatus is surrounded by a ring (FIG. 42), inturn made up of two concentric rings of electrodes in positive phase,followed by a ring of electrodes in positive phase alternating withdummy electrodes (for example floating electrodes), which are in turnfollowed by two rings of electrodes in positive phase. The dummyelectrodes are aligned with the columns of the conveyors.

It should be noted that the embodiment of the invention just describedadvantageously enables combination of the simplicity of programming andmanagement (number of phases for control of the lanes downwards) withthe precision (possibility of carrying out complex manipulations of theparticles of interest inside the array, having the possibility ofintervening independently on each of the individual cages thatconstitute it).

Apparatus for Recognition and Counting of Particles

To each of the methods for manipulation of particles according to thepresent invention, both with homogeneous arrays and with parking cellsand lanes or even with just lanes a part for detection of the particlescan be added in order to distinguish, recognize, characterize, or countcells/particles. The distinction or recognition can be obtainedaccording to the known art in different ways:

1. distinguishing/recognizing different particles/cells that have thesame reactive behaviour to the impressed forces F, but that affectdifferently reading of the sensor; for example, particles with adifferent index of transparency affect differently reading of theintensity of light of a photodiode;

2. distinguishing/recognizing particles/cells that have a differentbehaviour to the impressed forces F, but the same behaviour for thesensor; for example, cells of different dimensions can have a differentrate of displacement, and it is hence possible to recognize them bymonitoring the time used to pass from one block (BLOCK_i,j) to theadjacent one (BLOCK_i,j+1);

3. distinguishing/recognizing particles/cells that have a differentreactive behaviour to the impressed forces F and in any case a differentbehaviour for the sensor.

Recognition can be combined with a method for counting cells obtained bycombining the effect of the forces (F), through which to position eachcell (or group of cells) in a point corresponding to an element of anarray of sensors, and the capacity of identifying the presence of eachcell (or group of cells) by means of said sensors. In this way it ispossible, in addition to recognizing, also to count the particles ofeach type.

In each embodiment of apparatus for the manipulation of particlesaccording to the present invention, both with homogeneous arrays andwith parking cells and lanes or also with just lanes, it is consequentlypossible to add a part for detection of the particles.

Different embodiments are possible so that the detection is made viaimpedance meter or optical sensors. Of particular interest is thepossibility of detecting the particles even without an active substrate,i.e., without transistors.

Apparatus for the Manipulation of Particles without Transistors withImpedance Meter Sensors

It is hence possible to monitor the perturbation imposed by the presenceof a particle on the electrical field that is created between adjacentelements of an array of electrodes for the purpose of individuating,quantifying and/or qualifying the presence of particles. In the case ofhomogeneous arrays, a measurement can be made of the presence of one (ormore) particles and possibly its (their) characterization by means ofmeasurement of the impedance between the paths normally used to carrythe row signals and column signals.

With reference to FIG. 5, it may be understood how the impedancebetween, for example, Vrow_i and Vcol_j is markedly affected by thepresence or absence and by the type of particles possibly entrapped inthe cage CAGE_i,j, and slightly affected by the possible presence ofparticles in surrounding cages.

A similar measurement can be made in the case of an apparatus with lanesand parking cells. With reference to FIG. 23, it may be understood howthe impedance between, for example, Vrow_i and Vcol_j, is markedlyaffected by the presence or absence and by the type of particlespossibly entrapped in the cage of the block BLOCK_i,j, and only slightlyaffected by the possible presence of particles in surrounding cages.

Of course, it is possible to add row and column paths specifically fordetection, without thus having to multiplex the actuation and thedetection.

FIG. 43 shows a reading scheme according to the present invention fordetecting the impedance of the individual intersections (Zcage_ij)between generic lines of row signals (Ri) and column signals (Cj),without undergoing the influence of the coupling between adjacent rows(Zrow) and columns (Zcol), which otherwise would render detectionimpossible, in so far as their value is typically dominant with respectto Zcage_ij. This reading scheme can be obtained with an electronicsystem with components external to the microfabricated chip, and hencecompatible with the use of substrates without transistors, but can alsobe integrated on the chip in the case where transistors are available.

An input stimulus (Vin), with zero mean value, is applied selectively toa row (Ri), enabling only its multiplexers MRi. The other rowmultiplexers MR1 . . . MRi−1, MRi+1 . . . MRm connect the remaining rowsto ground. Just one column (Cj) corresponding to the co-ordinate of theintersection impedance (Zcage_ij) to be measured, is multiplexed on thevirtual ground (Vvgnd) of a transimpedance amplifier, the output ofwhich (Vout) is inversely proportional to the unknown impedance:Vout=−Vin*Zr/Zcage_ij

Said output voltage (Vout) can hence be used to derive Zcage_ij, withVin and Zr known. The output Vout, in general, can be processed,possibly together with the input Vin, by a block for processing thesignal (PROC), of an analog or digital type, to produce one or moreadditional—analog or digital—outputs (OUT), representing the measurementof the impedance and hence of the presence or otherwise or also of thetype of particle in the measurement point.

By way of example, we cite the case where the input (Vin) is a sinusoidat a known frequency. In this case, by processing the output of theamplifier (Vout) together with Vin it is readily possible to obtain withknown techniques an accurate measurement of Zcage_ij. For example,techniques of filtering such as lock-in amplifier filtering can possiblybe used in the block for processing the signal (PROC). Once again by wayof example we cite the possibility of applying an input voltage (Vin)formed by the sum of a number of sinusoids at different frequencies. Onaccount of the superposition of the effects, by separating the spectralcomponents of the output voltage (Vout) using analog or digital filtersin the processing block (PROC), it is possible to detect simultaneously,at all the frequencies which make up the input (Vin), the impedance(Zcage_ij) of the cage addressed by the row and column multiplexers(MRi) (MCj).

To speed up the reading operation it is possible to read in parallel allthe columns, replicating the amplifier and the processing block for eachcolumn. In this case, it is not necessary to use any column multiplexers(MCj).

Method and Apparatus for the Detection of Particles with Impedance MeterSensors

According to the present invention, a detection apparatus can beprovided also independently of the use of the chip as actuator. In thiscase, it is generally possible to increase the spatial resolution ofdetection points (at the limit obtaining a resolution equal to the pitchof the top metallization), obtaining an impedance meter image of thesample that enables resolution of individual cells.

Particularly useful is the study of the morphology of tissues formed bycell clusters in order to evaluate the roughness, humidity or otherparameters useful for cosmetic applications or for dermatologicalstudies. In this case, the measurement of impedance does not entail theuse of forces and can be effected between adjacent electrodes arrangedin a regular way in a two-dimensional space by positioning the tissue incontact with the substrate on which the array of electrodes is located.

The subject of the present invention is an apparatus that implementsthis technique by means of an array of blocks of electrodes, eachconstituted by at least one electrode connected to row signals and atleast one electrode connected to column signals, such that the impedancebetween said electrodes can be evaluated by measuring the impedancebetween row and column. A possible particle located in the neighbourhoodof each row and column intersection can in this way be detected bymeasuring the impedance between the row and column.

By way of example that by no means limits the scope of the presentinvention, we provide a possible implementation of said apparatus whichis particularly useful when the rows are formed on a substrate (SUB)whilst the columns are formed on the cover (LID) facing and set at adistance from the first substrate, or vice versa. In this way, in fact,it is possible to provide parallel rectangular electrodes equal to theentire length of the apparatus arranged horizontally on the substrate(SUB), to obtain row signals, and arranged on the cover (LID), to obtaincolumn signals. In this way, the measurement is made by evaluating theimpedance between each row and column in order to determine the presenceof a particle set between the row electrode and the column electrode atthe intersection between the two signals. The resulting apparatus can beobtained with just one level of metallization on the substrate (SUB) andone level of metallization on the cover (LID).

Apparatus for the Manipulation of Particles without Transistors withOptical Sensors and Transparent Electrodes

A further possibility of detection of the particles is constituted bythe use of optical sensors underneath the device, combined to the use oftransparent electrodes (such as Indium Tin Oxide—ITO). In this case,when the device is illuminated from above, the particles are detected bythe variations of optical power incident on the external detectionarray, underneath the device. As illustrated in FIG. 44, the underlyingdetection system can be constituted by an array of optical sensors(pixel), for example photodiodes or CCDs, in which the distance betweenadjacent elements of the array of sensors is 1/N times the distancebetween two adjacent blocks (BLOCK_i,j), with N=1 integer. The maincharacteristic of this technique lies in the possibility of aligning theparticles to be detected with the elements (pixel) of the sensor,improving the sensitivity of the measurements and obtaining a biuniquecorrespondence between particle and sensor element. This techniqueguarantees in fact that each particle can be located only andexclusively in the sensor area of just one element of the array ofsensors.

As an alternative, it is possible to use an array of external sensorsset at a distance from the actuation device, in which the lightreflected from above or transmitted from beneath is conveyed and focusedby a series of lenses towards the sensor, the elements (pixel) of whichare, however, aligned optically with the blocks of the array.

Apparatus for the Manipulation of Particles without Transistors withOptical Sensors and Non-Transparent Electrodes

A further possibility of detection of the particles is constituted bythe use of optical sensors (OPTISENS) underneath the device, combinedwith the use of non-transparent electrodes. In this case, the potentialholes (CAGE) can be obtained in the proximity of the substrate, in theregions not coated with the metal of the electrodes. Shown as aparticular case in FIG. 44 is a simple embodiment of the apparatusforming the subject of the present invention, in which the array ofelectrodes (EL) is constituted by just one electrode in the form of asquare grid (other geometrical shapes are obviously possible, such asrectangles, circles, hexagons, or triangles). In this case, blocks(BLOCK_i,j) are obtained, constituted (FIG. 44a ) by regions not coatedwith the metal of the electrode, where points of stable equilibrium(CAGE_i,j) are provided. In this way, if the substrate is transparent,it is possible to apply, underneath the apparatus (FIG. 44b ), a sensor(OPTISENS) constituted by an array of photosensitive elements (pixel)for the detection of the presence of entrapped particles in each of thepoints of stable equilibrium. In this connection, it is preferable forthe elements (pixel) of the array of sensors to be aligned opticallywith the array of points of stable equilibrium (CAGE_i,j), in which thedistance between adjacent elements of the array of sensors is 1/N timesthe distance between two adjacent blocks (BLOCK_i,j), with N=1 integer.This apparatus is particularly useful for counting the particlescontained in a liquid sample. In this case, the embodiment is limited tothe alignment of the particles (BEAD) with the elements (pixel) of thearray of sensors.

Given in FIG. 45, purely by way of example that in no way limits thescope of the present invention, are the results of an experimentconducted by means of a prototype obtained from a transparent-glasssubstrate with an electrode constituted by: a metal grid supplied with asinusoidal signal (Vphip); a cover lid, the bottom face of which isconductive and transparent (supplied with a signal in phase oppositionVphin); an external sensor, which detects the light collected by thebottom part of the device by means of the lenses of a microscope; and alight source that irradiates the device from above. In this case,optically associated to each element of the array of points of stableequilibrium (CAGE_i,j) is a multiplicity of pixels of the sensor. In thecase where an external sensor is used, as described previously it is notindispensable for the substrate (SUB) to be transparent since it ispossible to use the image collected from above, irradiating the devicewith reflected light.

With reference to the bottom part of FIG. 46, the signal (LINT) comingfrom each pixel of the sensor is converted into a digital signal bymeans of a hardware/software comparator that compares the signal comingfrom the sensor with a threshold (LLINE) appropriately fixed such thatthe logic value LDIG=0 (black) corresponds to the absence of anyparticle in the cell (BLOCK_i,j), whereas the logic value LDIG=1 (white)corresponds to the presence of a particle in the cell (BLOCK_i,j).Illustrated in FIG. 45a is an enlarged image of the device, in which theblocks (BLOCK_i,j) and the microspheres (BEAD) entrapped in the pointsof stable equilibrium (CAGE_i,j) are clearly visible, whilst illustratedin FIG. 45b is the processed signal corresponding to the same portion ofdevice. In the example shown, the processing consists in an inversion ofthe levels of grey, followed by a blurring and a thresholding. From theresulting image an automatic count may be readily obtained. Similarresults can be obtained using a contact sensor that gathers the lightfrom beneath, as illustrated in FIG. 46, or integrated within thesubstrate itself. The advantage of the use of contact sensors lies inthe fact that the use of the lenses of a microscope is not required. Theresult is an apparatus of reduced dimensions and hence portable.

It is evident to persons with ordinary skill in the sector that manyother possibilities exist of integration of sensors, which are generallyalso simpler if it is possible to use an active substrate withtransistors, which can be used for coupling an array of optical andimpedance meter sensors to the attraction cages.

In order to improve the performance due to the use of optical sensorsmicrolenses (MICROLENSE) can be used, which can for example be providedon the top part of the cover (LID) for conveying the light onto theentrapped particle. Illustrated in FIG. 47 is an example of this idea,in which it is shown schematically how the use of microlenses canimprove the sensitivity of the measurement (gathering the light thatotherwise would end up outside the sensitive region) and increase thecontrast between the different levels of signal associated to thepresence or absence of a particle (conveying all the rays of light intothe centre of force of the cage, where the particle is positioned). Itis moreover evident to persons skilled in the sector that the effects oflenses, parabolic dishes, prisms, minors, filters or polarizers can becombined for irradiating the apparatus.

As an alternative to the use of a two-dimensional array of opticalsensors (pixel) it is possible to use (FIG. 48) a one-dimensional array(SENSHEAD) with sensor elements (pixel) aligned optically with a row (orwith a column) of the array of points of stable equilibrium (CAGE_i,j),in which the distance between adjacent elements of the array of sensorsis 1/N times the distance between two adjacent blocks (BLOCK_i,j) on thesame row (or column), with N=1 integer. To acquire information on thepresence/absence of particles on the whole array, an acquisition iseffected in time sequence for each row (or column) of the array,displacing by a pitch (PITCH), after each acquisition, the array ofsensors (pixel) with respect to the array of blocks or vice versa in thedirection (HEADIR) parallel to the columns (or to the rows).

Shown in FIG. 48 purely by way of example that in no way limits thescope of the present invention is a possible embodiment of this idea. Inthis case, it is the device that moves, whilst the sensor (SENSHEAD),the light condenser (CONDENSOR), the precision optics (OPTIC), possiblefilters (FILTER) and the light source (LSOURCE) remain fixed.

Finally, it is possible to use a single photosensitive element to carryout a scan in time sequence of the entire array. In this case, aftereach acquisition, a displacement of the sensor (SENSHEAD) is effected inthe direction parallel to the rows, by a distance equal to the pitchbetween elements of the row. Next, at the end of each row a displacementof the sensor is effected in the direction parallel to the columns, by adistance equal to the pitch between elements of the column. Then afurther row is scanned, proceeding in the same manner up to completionof the entire array.

Finally, it is evident that the acquisition method and/or apparatusesdescribed previously can be applied to all of the methods and/orapparatuses forming the subject of the present invention, which isparticularly useful when the use of sensors is combined with themanipulation of particles or cells.

Aspects of the Disclosure

-   Aspect 1. A method for the manipulation of particles (BEAD) by means    of an at least two-dimensional array of groups (BLOCK_i,j) of    electrodes, comprising the steps of:-   i. generating a first configuration of field of force (F_i), which    presents at least one first point (CAGE_i,j) and at least one second    point (CAGE_i,j+1) of stable equilibrium for said particles (BEAD),    said points being positioned, respectively, on a first group    (BLOCK_i,j) and on a second group (BLOCK_i,j+1) of said array    immediately adjacent to the first, and being such that at least one    particle (BEAD) is entrapped in said first point of stable    equilibrium (CAGE_i,j);-   ii. generating at least one second configuration of field of force    (F_ii) such that a particle (BEAD) is pushed within a basin of    attraction of said at least one second point of stable equilibrium    (CAGE_i,j+1); and-   iii. generating again said first configuration of field of force    (F_i), such that said particle (BEAD) is attracted towards the    second point of stable equilibrium (CAGE_i,j+1); said method being    characterized in that said first configuration (F_i) and said second    configuration (F_ii) of field of force are generated by means of at    least two different configurations of first voltages (Vrow_i[p] and    Vcol_j[q]) applied to the electrodes of the first group of the array    (BLOCK_i,j) and of second voltages (Vrow_i[p] and Vcol_j+1[q])    applied to the electrodes of the second group of the array    (BLOCK_i,j+1).-   Aspect 2. The method according to aspect 1, comprising an iterative    execution of steps i) to iii) on a plurality of said groups of    electrodes of said array arranged adjacent to one another two by two    so as to displace at least one particle (BEAD) present in at least    one of said groups of electrodes (BLOCK_i,j) along paths constituted    by a succession of said adjacent groups of electrodes.-   Aspect 3. The method according to aspect 1, characterized in that    each group of electrodes (BLOCK_i,j) of the array is constituted by:    at least one first set of electrodes connected to voltages (Vphip,    Vphin) by means of row signals (Vrow_i[p]); at least one second set    of electrodes connected to voltages (Vphip, Vphin) by means of    column signals (Vcol_j[q]); and at least one electrode connected to    voltages (Vphip, Vphin) by means of a signal (Vcore) common to all    the groups of electrodes of the array and such that each pair of    adjacent groups of electrodes (BLOCK_i,j and BLOCK_i,j+1) can assume    said at least one second configuration of field of force (F_ii)    whilst all the other groups of electrodes of the array maintain said    first configuration (F_i), by means of modification of the voltages    applied via said row signals (Vrow_i[p]) and column signals    (Vcol_j[q]) connected to said pair of adjacent groups (BLOCK_i,j and    BLOCK_i,j+1) of electrodes.-   Aspect 4. The method according to aspect 1, characterized in that    each group of electrodes (BLOCK_i,j) of the array is constituted by    at least one first set of electrodes connected to voltages (Vphip,    Vphin) by means of electronic circuits (MUX_i,j and AND_i,j)    controlled by means of a first set of digital row signals (row_i)    and column signals (col_j) and by at least one electrode connected    to voltages (Vphip, Vphin) by means of a second set of signals    (Vcore) common to all the groups of electrodes (BLOCK_i,j) of the    array such that each pair of adjacent groups of electrodes    (BLOCK_i,j and BLOCK_i,j+1) can assume said second configuration of    field of force (F ii) whilst all the other groups of electrodes of    the array maintain said first configuration (F_i), by means of    modification of the voltages applied to said pair of adjacent groups    (BLOCK_i,j and BLOCK_i,j+1) of electrodes via said first set of row    signals (row_i) and column signals (col_j), which are connected to    the electronic circuits (MUX_i,j and AND_i,j) of said groups    (BLOCK_i,j and BLOCK_i,j+1) of electrodes.-   Aspect 5. A method for the manipulation of particles (BEAD) by means    of an array of first groups of electrodes that form at least one    lane (C_STORE; VRCHJ), comprising the steps of:-   i. generating a first configuration of field of force designed to    create at least one point of stable equilibrium (CAGE_i,j) for said    particles (BEAD), said point being positioned on at least one lane    (C_STORE; VRCHJ) and being such that at least one particle (BEAD) is    entrapped in said at least one point of stable equilibrium    (CAGE_i,j); and-   ii. displacing by one or more positions, each defined by at least    one electrode or by a first group of electrodes, all of said points    of stable equilibrium previously generated along said at least one    lane (C_STORE; VRCHJ);-   said method being characterized in that the points of stable    equilibrium present on said lanes are generated and moved by means    of the application to the electrodes of the first groups of    electrodes forming said at least one lane (C_STORE; VRCHJ) of at    least three different configurations of voltages (V1_j, V2_j, V3_j).-   Aspect 6. The method according to aspect 5, characterized in that it    envisages manipulation of said particles (BEAD) by means of a    plurality of lanes and by means of second groups of electrodes of    said array that form parking cells (BLOCK_i,j) arranged alongside    one another and/or alongside said lanes, said method comprising the    steps of:-   i. generating a second configuration of field of force designed to    create at least one point of stable equilibrium (CAGE_i,j) for said    particles (BEAD), which is positioned on a parking cell (BLOCK_i,j)    and is such that at least one particle (BEAD) is entrapped in said    at least one point of stable equilibrium (CAGE_i,j);-   ii. generating a third configuration of field of force such that a    particle (BEAD) entrapped in a parking cell (BLOCK_i,j) can be    pushed into a basin of attraction of a point of stable equilibrium    adjacent to the parking cell and formed by means of the electrodes    of said lanes (VRCHJ); and iii. displacing by one or more positions    all the points of stable equilibrium present in said lanes (VRCHJ)    along them;-   where the points of stable equilibrium of said lanes are generated    and moved by means of at least three different configurations of    voltages (V1_j, V2_j, V3_j) applied to the electrodes of said lanes    (VRCHJ); and in which the different field configurations for pushing    a particle (BEAD) from the point of stable equilibrium of one    parking cell (BLOCK_i,j) to one of the points of stable equilibrium    of the lanes (VRCHJ) or vice versa are generated by means of row    voltages (Vrow_i) and column voltages (Vcol_j and Vcage_j) applied    to the electrodes of the second groups (BLOCK_i,j) and by means of    voltages (V1_j, V2_j, V3_j) applied to the electrodes of the first    groups of electrodes forming said lanes (VRCHJ).-   Aspect 7. The method according to aspect 6, comprising the further    step of: generating a fourth configuration of field of force such    that a said particle (BEAD) can be pushed from a lane into a basin    of attraction (CAGE_i+1) of a point of stable equilibrium belonging    to a parking cell (BLOCK_i+1,j) different from the one in which said    particle was before being displaced on the lane.-   Aspect 8. The method according to aspect 7, characterized in that    said movements of points of stable equilibrium and said field    configurations necessary for pushing the particle (BEAD) from the    point of stable equilibrium of a parking cell (BLOCK_i,j) to one of    the points of stable equilibrium of the lanes (VRCHJ) and vice versa    act on any number of particles simultaneously, to displace each    particle along a different path.-   Aspect 9. The method according to aspect 5, characterized in that    for the manipulation of said particles (BEAD) by means of said array    of first groups of electrodes, the latter are pre-arranged for    providing at least two lanes (C_STORE and C_TEMP); and in that it    comprises the steps of:-   i. generating at least one point of stable equilibrium (CAGE_i,j)    for said particles (BEAD), which is positioned on at least one first    lane (C_STORE) and is such that at least one particle (BEAD) is    entrapped in said at least one point of stable equilibrium    (CAGE_i,j);-   ii. displacing by one or more positions all the points of stable    equilibrium along one or more lanes (C_STORE) so that said at least    one point of stable equilibrium (CAGE_i,j) can be shared by at least    one second lane (C_TEMP); and-   iii. displacing by one or more positions all the points of stable    equilibrium along one or more lanes (C_STORE) so that said particle    (BEAD) is entrapped in at least one point of stable equilibrium    (CAGE_i,j) belonging to said at least one second lane (C_TEMP); in    which said points of stable equilibrium of said lanes are generated    and moved by applying to the electrodes of said first groups of    electrodes of the array at least three different configurations of    voltages (V1_j, V2_j, V3_j) for each of said lanes.-   Aspect 10. The method according to aspect 1, characterized in that    it comprises, in sequence, a plurality of steps of entrapping of    said particles in points of stable equilibrium and of displacement    of said points of stable equilibrium combined in such a way as to    select one or more particles (BEAD).-   Aspect 11. The method according to aspect 1, characterized in that    it comprises, in sequence, a plurality of steps of entrapping of    said particles in points of stable equilibrium and of displacement    of said points of stable equilibrium combined in such a way as to    reorder the arrangement of two or more particles (BEAD).-   Aspect 12. The method according to aspect 1, characterized in that    it comprises, in sequence, a plurality of steps of entrapping of    said particles in points of stable equilibrium and of displacement    of said points of stable equilibrium combined in such a way as to    displace one or more particles (BEAD) present on one and the same    group of electrodes (BLOCK_i,j).-   Aspect 13. The method according to aspect 1, characterized in that    it comprises, in sequence, a plurality of steps of entrapping of    said particles in points of stable equilibrium and of displacement    of said points of stable equilibrium combined in such a way as to    separate and/or move away two or more particles (BEAD) positioned on    one and the same group of electrodes (BLOCK_i,j) towards at least    two different positions.-   Aspect 14. The method according to aspect 1, characterized in that    said field of force (F) comprises at least one of the following    forces:-   i. positive dielectrophoresis (PDEP);-   ii. negative dielectrophoresis (NDEP);-   iii. electrophoresis (EF);-   iv. electrohydrodynamic flows (EHD); and-   v. electrowetting on dielectric (EWOD)-   Aspect 15. A method for detection and/or characterization and/or    quantification and/or recognition of cells or particles (BEAD) in    aqueous suspension or aggregated in tissues comprising a measurement    of impedance between at least one first electrode connected to at    least one first row signal and at least one second electrode    adjacent to said first electrode, said second electrode being    connected to at least one first column signal, said method being    characterized in that said measurement between said at least two    adjacent electrodes is made by evaluating the impedance between said    first row signal and said first column signal.-   Aspect 16. The method according to aspect, 15 characterized in that    it is carried out further a detection and/or characterization and/or    quantification and/or recognition of cells or particles (BEAD) in    aqueous suspension comprising a measurement of impedance between at    least one first electrode connected to at least one first row signal    and at least one second electrode adjacent to said first electrode,    said second electrode being connected to at least one first column    signal, said method being characterized in that said measurement    between said at least two adjacent electrodes is made by evaluating    the impedance between said first row signal and said first column    signal.-   Aspect 17. An apparatus for the manipulation of particles (BEAD),    characterized in that it comprises:-   i. an array of electrodes forming an array of blocks of groups    (BLOCK_i,j) of electrodes arranged in rows and columns, each block    comprising: a first group of electrodes connected to column signals    (Vcol_j[q]) common to all the first groups of the same column of    blocks; a second group of electrodes connected to row signals    (Vrow_i[p]) common to all the second groups of the same row of    blocks; and a third group of electrodes connected to signals common    to all the blocks of groups of electrodes (BLOCK_i,j) of the array    of electrodes;-   ii. means for generating at least two different voltages (Vphin,    Vphip); and iii. means for distributing said voltages (Vphin, Vphip)    to said row signals (Vrow_i[p]) and to said column signals    (Vcol_j[q]) and to said common signals;    in which said means for distributing said voltages (Vphin, Vphip)    are such that for each pair formed by a first block (BLOCK_i,j) of    groups of electrodes and by a second block (BLOCK_i,j+1) of groups    of electrodes adjacent to the first block, at least one first    configuration (F_i) and one second configuration (F_ii) of field of    force are generated obtained by applying two different    configurations of said two voltages (Vphin, Vphip) to the row    signals (Vrow_i[p]) and column signals (Vcol_j[q] and Vcol_j+1[q]);    said first configuration (F_i) of field of force being designed to    create at least one first point (CAGE_i,j) and at least one second    point (CAGE_i,j+1) of stable equilibrium for said particles (BEAD),    said points being positioned, respectively, on said first block    (BLOCK_i,j) and on said second block (BLOCK_i,j+1) of groups of    electrodes of the array; and said second configuration (F_ii) of    field of force being designed to push said particle (BEAD) possibly    entrapped in said first point of stable equilibrium (CAGE_i,j) into    a basin of attraction of said at least one second point of stable    equilibrium (CAGE_i,j+1).-   Aspect 18. The apparatus according to aspect 17, characterized in    that said first group of electrodes of each said block (BLOCK_i,j)    comprises at least one first electrode (ring_i,j_1) connected to a    column signal (Vcol_j) common to all the groups of the same column;    said second group of electrodes of each block comprises at least one    second electrode (ring_i,j_2) connected to a row signal (Vrow_i)    common to all the groups of the same row; and said third group of    electrodes of each block comprises at least one third electrode    (EL_i,j) connected to a common signal (Vcore), in which said third    electrode (EL_i,j) is surrounded by said first electrode    (ring_i,j_1) and said third electrode (EL_i,j) and said first    electrode (ring_i,j_1) are surrounded by said second electrode    (ring_i,j_2);-   Aspect 19. The apparatus according to aspect 17, characterized in    that said first group of electrodes of each said block (BLOCK_i,j)    comprises at least one first electrode (elle_j) connected to a    column signal (Venable_j) common to all the groups of the same    column; said second group of electrodes of each block comprises at    least one second electrode (wallx_i) connected to a row signal    (Vrow_i[x]) common to all the groups of the same row; and said third    group of electrodes of each block comprises at least one third    electrode (EL_i,j) connected to a common signal (Vcore); in which    said second group of electrodes of each block moreover comprises at    least one fourth electrode (wally_i) connected to a row signal    (Vrow_i[y]) common to all the groups of the same row; and where said    third electrode (EL_i,j) is flanked in two directions adjacent by    said first electrode (elle_j), and said first electrode (elle_j) is    flanked by said second electrode (wallx_i) and said fourth electrode    (wally_i).-   Aspect 20. The apparatus according to aspect 17, characterized in    that said first group of electrodes of each said block (BLOCK_i,j)    is distinguished into electrodes with even column index and odd    column index, whilst said second group of electrodes of each said    block (BLOCK_i,j) is distinguished into electrodes with even row    index and odd row index; in which said third group of each block    comprises at least one third electrode (EL_i,j) connected to a    common signal (Vcore); and in which said first group comprises at    least one first electrode connected to a column signal (Vcol_i[D1])    common to the blocks of the same column with even column index or to    a column signal (Vcol_i[D2]) common to the blocks of the same column    with odd column index; and at least one second electrode connected    to a column signal (Vcol_i[U1]) common to the blocks of the same    column with even column index or to a column signal (Vcol_i[U2])    common to the blocks of the same column with odd column index; and    in which said second group comprises at least one fourth electrode    connected to a row signal (Vrow_i[R1]) common to the blocks of the    same row with even row index or to a row signal (Vrow_i[R2]) common    to the blocks of the same row with odd row index and at least a    fifth electrode connected to a row signal (Vrow_i[L1]) common to the    blocks of the same row with even row index or to a row signal    (Vrow_i[L2]) common to the blocks of the same row with odd row    index.-   Aspect 21. An apparatus for the manipulation of particles (BEAD),    comprising:-   i. an array of electrodes forming an array of blocks of groups    (BLOCK_i,j) of electrodes arranged in rows and columns, each block    comprising: a first group of at least one first electrode (EL_i,j)    connected to a signal common to all the blocks; and a second group    of at least one second electrode (ring_i,j) connected to an output    signal of a circuit (MUX_i,j), driven by at least one second circuit    (AND_i,j);-   ii. means for generating at least two different voltages (Vphin,    Vphip); and-   iii. means generating row signals (row_i) common to all the blocks    (BLOCK_i,j) of the same row and column signals (col_j) common to all    the blocks (BLOCK_i,j) of the same column for driving said circuits    (AND_i,j) and by means of which the voltage (Vphin, Vphip) to    connect to said at least one second electrode (ring_i,j) of each    block (BLOCK_i,j) is selected;-   where for each pair formed by a first block (BLOCK_i,j) and a second    block (BLOCK_i,j+1) adjacent to the first block said means for    generation of signals determine the creation of at least one first    configuration (F_i) and one second configuration (F_ii) of field of    force by applying two different configurations of values to the row    signals (Vrow_i) and column signals (Vcol_j and Vcol_j+1) such that    said first configuration (F_i) of field of force presents at least    one first point (CAGE_i,j) of stable equilibrium and at least one    second point (CAGE_i,j+1) of stable equilibrium for said particles    (BEAD), said points being positioned, respectively, on said first    block (BLOCK_i,j) and on said second block (BLOCK_i,j+1) of the    array, and being such that said second configuration (F_ii) of field    of force is designed to push said particle (BEAD) that is possibly    entrapped in said first point of stable equilibrium (CAGE_i,j) into    a basin of attraction of said at least one second point of stable    equilibrium (CAGE_i,j+1).-   Aspect 22. The apparatus according to aspect 21, characterized in    that said first group of electrodes of each said block (BLOCK_i,j)    comprises at least one first electrode (EL_i,j) connected to a    signal common to all the blocks constituted by one (Vphin) of said    different voltages; and in that said second group of electrodes    comprises at least one second electrode (ring_i,j) connected to the    output signal of a circuit that constitutes a deviator (MUX_i,j),    such that just one of two different signals (Vphin, Vphip) at input    to the deviator can be connected at output from the deviator    according to the value of the output signal of a further circuit    (AND_i,j), which performs a logic function between the values of the    row signals (row_i) and column signals (col_j); and in which said    first electrode (EL_i,j) is surrounded by said second electrode    (ring_i,j).-   Aspect 23. An apparatus for the manipulation of particles (BEAD),    comprising:-   i. an array of electrodes comprising first groups of electrodes,    each of which is constituted by at least one first electrode    connected to a first signal (V1_j; S1), by at least one second    electrode connected to a second signal (V2_j; S2) and by at least    one third electrode connected to a third signal (V3_j; S3) such that    the set of said first groups of electrodes forms at least one first    lane (VRCHJ; C_STORE), designed to move said particles (BEAD) in a    chosen direction;-   ii. means for generating at least two different voltages (Vphin,    Vphip); and-   iii. means for distributing said voltages (Vphin, Vphip) to said at    least one first signal (V1_j; S1), second signal (V2_j; S2) and    third signal (V3_j; S2);-   so that each lane (VRCHJ; C_STORE) can generate at least one first    configuration (F_i) of field of force designed to create at least    one first point of stable equilibrium (CAGE_i,j) for said particles    (BEAD), which is positioned on said at least one first lane (VRCHJ;    C_STORE) and is such that at least one particle (BEAD) is entrapped    in said at least one point of stable equilibrium (CAGE_i,j) and can    be displaced along said first lane (VRCHJ; C_STORE), simultaneously    displacing all of said first points of stable equilibrium present on    the first lane by applying to the electrodes of said first groups of    electrodes at least three different configurations of voltages on    said signals (V1_j, V2_j, V3_j; S1, S2, S3).-   Aspect 24. The apparatus according to aspect 23, characterized in    that said array of electrodes moreover comprises an array of blocks    (BLOCK_i,j) of groups of electrodes arranged in rows and columns for    the manipulation of said particles (BEAD), each block (BLOCK_i,j) of    the array comprising:-   i. a second group of electrodes of said array connected to column    signals (Vcage_j, Vcol_j) common to all the blocks (BLOCK_i,j) of    the same column;-   ii. a third group of electrodes of said array connected to row    signals (Vrow_i) common to all the groups of the same row; and-   iii. and a fourth group of electrodes of said array connected to    signals (Vp_j) common to all the blocks (BLOCK_i,j);-   such that each block (BLOCK_i,j) can constitute a parking cell    (CAGE_i,j) for said particles arranged alongside one another and/or    alongside said at least one first lane;-   said apparatus comprising moreover means for distributing said    voltages (Vphin, Vphip) to said row signals (Vrow_i) and to said    column signals (Vcage_j, Vcol_j) and to said common signals (Vp_j)    so that each block (BLOCK_i,j) of the array can generate at least    one first configuration (F_i) and one second configuration (F_ii) of    field of force by applying two different configurations of voltages    (Vphin, Vphip) to the row signals (Vrow_i) and column signals    (Vcage_j, Vcol_j) such that said first configuration (F_i) of field    of force presents at least one second point of stable equilibrium    (CAGE_i,j) for said particles (BEAD) positioned on said block    (BLOCK_i,j) and such that said second configuration (F_ii) of field    of force pushes said particle (BEAD) into a basin of attraction of    said at least one first point of stable equilibrium formed by means    of the electrodes of said at least one first lane (VRCHJ) and    displaceable along said lane (VRCH_j).-   Aspect 25. The apparatus according to aspect 24, characterized in    that said means for distributing said voltages (Vphin, Vphip) to    said row signals (Vrow_i) and/or to said column signals (Vcage_j,    Vcol_j) and/or to said common signals (Vp_j) are constituted by    signal-conditioning circuits and/or memory elements.-   Aspect 26. The apparatus according to aspect 25, characterized in    that said means for distributing said voltages (Vphin, Vphip) to    said at least one first signal (V1_j), one second signal (V2_j), and    one third signal (V3_j) are constituted by signal-conditioning    circuits and/or memory elements.-   Aspect 27. The apparatus according to aspect 23, characterized in    that said array of electrodes moreover comprises:-   i. second groups of electrodes, each of which is constituted by at    least one first electrode connected to a fourth signal (T1), by at    least one second electrode connected to a fifth signal (T2), and by    at least one third electrode connected to a sixth signal (T3) such    that the set of said second groups of electrodes forms at least one    second lane (C_TMP), designed to move said particles (BEAD) in a    chosen direction;-   ii. at least one point of exchange formed by means of adjacent    electrodes belonging to said first and, respectively, said second at    least one lane; and-   iii. means for distributing said voltages (Vphin, Vphip) to said at    least one fourth signal (T1), fifth signal (T2) and sixth signal    (T3);-   so that each at least one first and second lanes (C_STORE and    C_TEMP) is designed to generate selectively at least one first    configuration (F_i) and one second configuration (F_ii) of field of    force such that said first configuration (F_i) of field of force    presents at least one first and one second point of stable    equilibrium on said first and second lane (C_STORE or C_TEMP),    respectively, said points being such that at least one particle    (BEAD) is entrapped in said at least one first or second point of    stable equilibrium and can be displaced along said lanes (C_STORE or    C_TEMP), simultaneously displacing all of said first or second    points of stable equilibrium present on the first or second lane by    applying to the electrodes of said first and second groups of    electrodes at least three different configurations of voltages on    said signals (S1 and/or S2 and/or S3 and/or T1 and/or T2 and/or T3)    and such that said second configuration (F_ii) of field of force is    formed at said point of exchange and is designed to push said    particle (BEAD) into a basin of attraction of said at least one    second point of stable equilibrium obtained by means of the    electrodes of the second lane (C_TEMP).-   Aspect 28. The apparatus according to aspect 27, characterized in    that it comprises at least one first microchamber (MCH) comprising:    one or more first lanes (VC_1), driven by at least three voltages    (V1, V2 and V3), and at least one second (HCONV) lane, driven by at    least three signals (H1, H2 and H3) with at least one first point of    exchange between said first lane (VC_1) and said second lane    (HCONV); and at least one second microchamber (RCH); characterized    in that it comprises at least one third lane (RCONV), driven by at    least three signals (R1, R2 and R3) and at least one second point of    exchange between said second lane (HCONV) and said third lane    (RCONV) such that it is possible to bring a particle from said first    microchamber (MCH) into said second microchamber (RCH) through a    said first (VC_1) and said second lane (HCONV), and said at least    one first and at least one second point of exchange.-   Aspect 29. The apparatus according to aspect 27, characterized in    that it comprises at least one first microchamber (MCH) comprising    at least one first lane (VC_1), driven by at least three signals    (V1, V2 and V3) and at least one second (HCONV) lane driven by at    least three signals (H1, H2 and H3) with a first point of exchange    between said first lane (VC_1) and said second lane (HCONV), and at    least one second microchamber (RCH); characterized in that it    comprises at least one third lane (RCONV) synchronous with said    first lane and driven by said at least three signals (V1, V2 and V3)    and at least one second point of exchange between said second lane    (HCONV) and said third lane (RCONV) obtained by means of an    electrode driven by a signal (THR) such that it is possible to bring    a particle from said first microchamber (MCH) into said second    microchamber (RCH) through said first lane (VC_1) and said second    lane (HCONV) and said first and said second points of exchange.-   Aspect 30. The apparatus according to aspect 27, characterized in    that said means for distributing said voltages (Vphin, Vphip) to    said signals are obtained by means of signal-conditioning circuits    and/or memory elements.-   Aspect 31. The apparatus according to aspect 27, characterized in    that it is divided by a diaphragm (CHW) made of polymeric material    into two microchambers (MCH, RCH), a first microchamber (MCH)    comprising:-   a. a first multiplicity and a second multiplicity of lanes forming a    vertical closed loop (VC1 _(—1) . . . VC1_NCV and VC2_1 . . .    VC2_NCV), designed to form each a plurality (NI) of said points of    stable equilibrium (CAGES) for entrapping said particles (BEAD), by    means of three phases (V1, V2 and V3), connected repeatedly at    respective iterations (I_1 . . . I_NI) of groups of three electrodes    on each lane;-   b. a first horizontal lane and a second horizontal lane (HCONV_UP,    HCONV_DOWN), respectively, an upper one and a lower one, driven by    four phases (H1, H2, H3 and H4) and comprising a plurality (NCV) of    points of exchange with the vertical lanes, active in one of said    phases (V2+H3), so that it will be possible to transfer    simultaneously the contents of at least one point of stable    equilibrium from the vertical lanes to the first horizontal lane or    second horizontal lane;-   c. a third horizontal lane (HCONV_AUX), driven by four phases (AUX1,    AUX2, AUX3 and AUX4), which comprises a plurality (NCAUX1) of points    of exchange with the upper horizontal lane (HCONV_UP) and an    identical number (NCAUX2) of points of exchange with the lower    horizontal lane (HCONV_DOWN), situated in positions corresponding to    one another;-   d. a completely programmable array of electrodes designed to form in    use an array of individually programmable attraction cages for said    particles defined by points of stable equilibrium of a field of    force generated via said electrodes;-   e. a first vertical dump lane and a second vertical dump lane    (VCW_UP and VCW_DOWN), driven by three phases in a way substantially    similar to said vertical lanes (VC1_i and VC2_j), which have the    function of removing undesired particles from the array; and-   f. a vertical long dump lane (VCW_LONG), having a dimension    approximately twice that of the other said vertical lanes (VC1_i and    VC2_j) set in a portion of said first microchamber (MCH) situated on    the opposite side with respect to the array.-   Aspect 32. The apparatus according to aspect 31, characterized in    that said second microchamber (RCH) comprises: an exit lane (RCONV),    driven by four phases (R1, R2, R3 and R4), for conveying particles    of interest leaving said array into the second microchamber, through    a discontinuity of said diaphragm made of polymeric material (CHW)    constituting a passage of communication between the two    microchambers; and a horizontal feedback lane (HCONV_FB), driven by    four phases (FB1, FB2, FB3 and FB4), lying substantially on one and    the same straight line identified by said auxiliary horizontal lane,    by means of which it is possible to bring a particle back from the    exit lane (RCONV), and hence from the second microchamber (RCH),    into the array, once again through the aforesaid passage in the    diaphragm (CHW).-   Aspect 33. The apparatus according to aspect 17, characterized in    that it comprises at least two microchambers and in that it presents    an arrangement of said electrodes of said array of electrodes such    that it is possible to displace said particles (BEAD) from one    microchamber to the other and vice versa by driving said electrodes    with appropriate signals.-   Aspect 34. The apparatus according to aspect 17, characterized in    that said electrodes are made on a substantially planar substrate    (SUB); and in that it comprises a further electrode (ITO) made on a    further substrate (LID) set at a distance from and facing said first    substrate (SUB), said further electrode (ITO) being electrically    connected to a further electrical signal.-   Aspect 35. An apparatus for detection and/or characterization and/or    quantification and/or recognition of cells or particles (BEAD) in    aqueous suspension or aggregated in tissues, comprising:-   i. an array of groups of electrodes comprising a first group of    electrodes connected to column signals (Cj) common to all the groups    of the same column and at least one second group of electrodes    connected to row signals (R1) common to all the groups of the same    row;-   ii. means for generating at least one voltage (Vin);-   iii. at least one circuit for reading the impedance (Zcage_i,j)    given by the intersection of a row (Ri) and a column (Cj); and-   iv. means (MRi) for distributing said voltage (Vin) to said row    signals (R1) and means (MCj) for distributing said column signals    (Cj) to said at least one read circuit;-   in which by connecting said read circuit to a column (Cj) and by    applying said voltage to a row, (Ri) the output signal (Vout and/or    out) of said read circuit is affected by the value of the impedance    (Zcage_ij) between that column and that row.-   Aspect 36. The apparatus according to aspect 35, characterized in    comprising means for reading the variation of impedance at one or    more potential holes (CAGE) due to the presence of one or more    particles (BEAD).-   Aspect 37. The apparatus according to aspect 36, characterized in    that said means for reading the variation of impedance are obtained    by means of row signals (R1) and column signals (Cj) such that at    least one or more particles (BEAD) alter the value of impedance due    to the intersection between at least one row (Ri) and at least one    column (Cj).-   Aspect 38. The apparatus according to aspect 37, characterized in    that said column signals (Cj) are obtained on a first substantially    planar substrate (SUB) and in that said row signals (R1) are    obtained on a further substrate (LID) set at a distance from and    facing said first substrate (SUB).-   Aspect 39. The apparatus according to aspect 36, characterized in    that said means for reading the variation of impedance are obtained    by means of the same signals used for the creation of the    distributions of said fields of force (F).-   Aspect 40. An apparatus for the manipulation of particles (BEAD)    comprising:-   i. means for generating at least one voltage (Vphin); and-   ii. at least one electrode (EL) connected to said electrical signal    (Vphin);-   said apparatus being characterized in that said at least one    electrode comprises holes such as to form a grid and, as a    consequence of the application of said electrical signal to said    electrode, points of stable equilibrium (CAGE_i,j) for said    particles (BEAD).-   Aspect 41. The apparatus for the manipulation and detection and/or    identification of particles (BEAD) according to aspect 40,    characterized in that it comprises means for reading the variation    in intensity of a light (LIGHT) reflected and/or transmitted to one    or more of said points of stable equilibrium due to the presence of    one or more particles (BEAD) entrapped therein.-   Aspect 42. The apparatus according to aspect 41, characterized in    that said means for reading the variation in intensity of the    reflected and/or transmitted light comprise an array of one or more    optical sensors (pixel) arranged on a mobile head (SENSHEAD) and    separate from the substrate (SUB) such that reading of the variation    of reflected and/or transmitted light (LIGHT) at each point of    equilibrium can be obtained by aligning said head with respect to    said point of equilibrium by means of a relative motion between head    and substrate.-   Aspect 43. The apparatus according to aspect 41, characterized in    that said means for reading the variation of the intensity of the    light reflected and/or transmitted comprise an array of one or more    optical sensors (pixel) integrated within said substrate (SUB).-   Aspect 44. The apparatus according to aspect 41, characterized in    that said means for reading the variation in intensity of the light    comprise an array of one or more external optical sensors (pixel)    positioned in a further substrate (OPTISENS) set underneath said    first substrate (SUB) or on top of said second substrate (LID).-   Aspect 45. The apparatus according to aspect 43, characterized in    that it comprises an array of lenses (MICROLENSES), each of which    concentrates the incident light in a position corresponding to each    electrode or group of electrodes (BLOCK_i,j) in which a said point    of stable equilibrium (CAGE_i,j) for said particles (BEAD) can be    provided.

The invention claimed is:
 1. A method for detection and/orcharacterization and/or quantification and/or recognition of cells orparticles (BEAD) in aqueous suspension or aggregated in tissuescomprising: measuring impedance between at least one first electrodeconnected to at least one first row signal and at least one secondelectrode diagonally adjacent to said first electrode, said secondelectrode being connected to at least one first column signal, saidmethod being characterized in that said measurement between said atleast two diagonally adjacent electrodes is made by evaluating theimpedance given by a diagonal intersection of said first row signal andsaid first column signal.
 2. The method according to claim 1, furthercomprising the step of carrying out a detection and/or characterizationand/or quantification and/or recognition of cells or particles inaqueous suspension using the measurement of impedance between at leastone first electrode connected to at least one first row signal and atleast one second electrode diagonally adjacent to said first electrode,said second electrode being connected to at least one first columnsignal, wherein said measurement between said at least two adjacentelectrodes is made by evaluating the impedance given by a diagonalintersection of said first row signal and said first column signal. 3.An apparatus for detection and/or characterization and/or quantificationand/or recognition of cells or particles (BEAD) in aqueous suspension oraggregated in tissues, comprising: i. an array of groups of electrodescomprising a first group of electrodes aligned in columns and connectedto column signals (Cj) common to all the groups of the same column andat least one second group of electrodes aligned in rows and connected torow signals (R1) common to all the groups of the same row; ii. at leastone circuit for reading the impedance (Zcage_i,j) given by an diagonalintersection of a row signal (Ri) and a column signal (Cj); and iii. afirst signal-conditioning circuit (MRi) for distributing at least onevoltage (Vin) to said row signals (Ri) and a second signal-conditioningcircuit (MCj) for distributing said column signals (Cj) to said at leastone read circuit; wherein iv. said read circuit is configured togenerate an output signal (Vout and/or out) which is affected by thevalue of the impedance (Zcage_ij) between a column (Cj) connected tosaid read circuit and a row (Ri) to which said voltage is applied. 4.The apparatus according to claim 3, further comprising a circuit forreading the variation of impedance at one or more potential holes (CAGE)due to the presence of one or more particles (BEAD).
 5. The apparatusaccording to claim 4, wherein said circuit for reading the variation ofimpedance at one or more potential holes is configured such that atleast one or more particles (BEAD) alter the value of impedance due tothe diagonal intersection between at least one row (Ri) and at least onecolumn (Cj) to which row signals (Ri) and column signals (Cj) areapplied.
 6. The apparatus according to claim 4, characterized in thatsaid circuit for reading the variation of impedance is configured toreceive the same signals used for the creation of the distributions ofsaid fields of force (F).
 7. The apparatus according to claim 3, whereinthe first signal-conditioning circuit and/or the second signalconditioning circuit form a multiplexer.
 8. An apparatus for detectionand/or characterization and/or quantification and/or recognition ofcells or particles (BEAD) in aqueous suspension or aggregated intissues, comprising: i. an array of groups of electrodes comprising afirst group of electrodes aligned in columns and connected to columnsignals (Cj) common to all the groups of the same column and at leastone second group of electrodes aligned in rows and connected to rowsignals (R1) common to all the groups of the same row; ii. at least onecircuit for reading the impedance (Zcage_i,j) given by an intersectionof a row (Ri) and a column (Cj); and iiii. a first signal-conditioningcircuit (MRi) for distributing at least one voltage (Vin) to said rowsignals (Ri) and a second signal-conditioning circuit (MCj) fordistributing said column signals (Cj) to said at least one read circuit;wherein iv. said read circuit is configured to generate an output signal(Vout and/or out) which is affected by the value of the impedance(Zcage_ij) between a column (Cj) connected to said read circuit and atrow (Ri) to which said voltage is applied; and v. said column signals(Cj) are obtained on a first substantially planar substrate (SUB) andsaid row signals (Ri) are obtained on a further substrate (LID) set at adistance from and facing said first substrate (SUB).